Chizuru Yukimura (雪村 千鶴 Yukimura Chizuru?)
Voiced by: Houko Kuwashima (Japanese); Brittney Karbowski (English)

Chizuru is the main protagonist of the series. She comes to Kyoto in search of her missing father, Kodo Yukimura (雪村 綱道 Yukimura Kōdō?), a practitioner of Western medicine. Unknown to Chizuru, her father under orders from the Shogunate develops the Water of Life (復ち水 Ochimizu?), an elixir that increases the drinker's healing abilities, speed, and strength yet could turn the drinker into a mindless killing monster called a Rasetsu.

At the beginning of the series, she is seen being chased by two men, who were suddenly attacked by two Rasetsu. She witnesses Hajime Saito kill off the Rasetsu and is then taken to the Shinsengumi headquarters by Toshizo Hijikata, where they allow her to stay, because they too were in search of her father. Eventually, she falls in love with Hijikata Toshizo.

Chizuru has a secret unknown to herself: she is in fact a pureblood member of the Oni Ichizoku (the Demon Clan), and the head of the Oni of the East is of Yukimura descent. Because Oni have scattered and declined in numbers over the centuries, Chikage Kazama seeks her out to create a lineage of pure-blooded Oni. She is the wielder of a short sword which is a mate to with Kaoru's katana. They are part of the Yukimura Family's heirloom.

Toshizo Hijikata (土方 歳三 Hijikata Toshizō?)
Voiced by: Shinichiro Miki (Japanese); Andrew Love (English)

Hijikata is the Vice-commander of the Shinsengumi and makes most of the Shinsengumi's decisions. He is called Oni-fukuchou (Demon Vice-commander) and is both feared and respected by his men. He is also often referred to as "Toshi" by Kondo. Hijikata is the one who brought Chizuru back to the Shinsengumi headquarters the night she witnessed a Rasetsu on the loose that attacked two men chasing her. He is responsible for her welfare, and even though he acts as if this were to his dismay, he cares for Chizuru's safety, and is often shown protecting her during battles.

Hijikata drinks the Water of Life and becomes a Rasetsu in order to defeat Chikage, fueled by rage after seeing fellow Shinsengumi warrior Genzaburo Inoue dead. Even though Inoue had been killed by Satchō warriors (Satsuma-Chōshū alliance), Hijikata takes it out on Chikage, starting a duel as Chikage goads him. At the end of Season 2 he acknowledge his love for Chizuru.

At the end of the anime series he either dies from a combination of the Water of Life and wounds that he attained through a battle with Chikage Kazama, or he lives (as suggested by the fact that his body didn't disappear). His ending is ambiguous. But hinted that he's still alive as wiping Chizuru's tear and saying her name while smiling at her. He is based on the historical Hijikata Toshizō.

Souji Okita (沖田 総司 Okita Sōji?)
Voiced by: Showtaro Morikubo (Japanese); Blake Shepard (English)

Okita is the First Division Captain and a peerless swordsman. He suffers from tuberculosis, and is later visited by Nagumo Kaoru who gives him a bottle of the Water of Life to cure his tuberculosis. Driven by guilt at being incapacitated by his illness, Okita drinks the elixir. Although he becomes a Rasetsu, the Shinsengumi discover that he does not benefit from accelerated healing when wounded by silver weapons, thus discovering the weakness of Rasetsu.

Souji was sent away because his illness got worse, but he later showed up to confront Hijikata about Kondo's death. He was last seen protecting Hijikata from rogue samurais, and turned into ashes after killing all the rogues. He is based on the historical Okita Sōji.

Isami Kondo (近藤 勇 Kondō Isami?)
Voiced by: Tōru Ōkawa (Japanese); David Wald (English)

Kondo is the Commander of the Shinsengumi. He is very emotional, but despite this, he has the trust, respect, and loyalty of his men. He is based on the historical Isami Kondō.

Keisuke Yamanami (山南 敬助 Yamanami Keisuke?)
Voiced by: Nobuo Tobita (Japanese); David Matranga (English)

Usually called "Sannan-san" (based on an alternate reading of his surname), he is Vice-commander of the Shinsengumi, along with Hijikata. Both a scholar and a ruthless tactician, he is kind and caring, albeit a bit reserved. After an injury which resulted in him being unable to use his left arm and thus wield a katana, he becomes cold and distant; he proceeds to research and improve on the Water of Life in secret in order to overcome its side effects, eventually drinking it and becoming one of the first few Rasetsu with self control, creating the Shinsengumi's Rasetsu Corps. He is based on the historical Yamanami Keisuke.

Shinpachi Nagakura (永倉 新八 Nagakura Shinpachi?)
Voiced by: Tomohiro Tsuboi (Japanese); Kalob Martinez (English)

Shinpachi is the Second Division captain of the Shinsengumi. He is usually very cheerful, especially when he is with his comrades. Shinpachi is especially close to Harada and together they often pick on Heisuke. He is very skilled with the sword, but he is also a borderline alcoholic and womanizer, as he is seen breaking the curfew one night to be out drinking. He is based on the historical Nagakura Shinpachi.

Sanosuke Harada (原田 左之助 Harada Sanosuke?)
Voiced by: Kōji Yusa (Japanese); Illich Guardiola (English)

Harada is the Tenth Division Captain and prefers the spear to the sword, but he is seen carrying both. In the anime he has a fierce rivalry with Kyō Shiranui, pitting his spear against Shiranui's pistol. He is based on the historical Harada Sanosuke.

Heisuke Todo
Voiced by: Hiroyuki Yoshino (Japanese); Greg Ayres (English)

Heisuke is the Eighth Division captain of the Shinsengumi. He is around the same age as Chizuru and is very protective of her, before he died. He leaves the Shinsengumi, following Itou along with Hajime Saito, but later returns. He too has taken the Water of Life to recover from injuries. He is based on the historical Tōdō Heisuke.

Hajime Saito (斎藤 一 Saitō Hajime?)
Voiced by: Kōsuke Toriumi (Japanese); Leraldo Anzaldua (English)

Saito is the leader of the Third Division who is a master of the left hand sword technique and is a skilled swordsman who evens Okita's skills. He is taciturn but also very loyal, polite, and wise. He often analyzes the situation before attacking to figure out which actions are necessary to complete the mission. Saito left the Shinsengumi apparently to join Itou's group, but later it turns out that he was commanded by Hijikata to infiltrate Itou's group for information concerning Itou's plans. In the anime he is seen making a final charge against the enemy as his comrades are all slain. He is based on the historical Saitō Hajime.

Kaoru Nagumo (南雲 薫 Nagumo Kaoru?)
Voiced by: Hasumi Itō (Japanese); Shannon Emerick (English)

First seen as a lovely dressed young woman who was saved by Okita from punks. Okita also notices that she resembles Chizuru, though Kaoru is more prettier, he is actually Chizuru's twin brother who was separated from her during childhood. He's made to dress as a woman but is seen as a boy near the end of the first season of the anime. He is one of the strongest Oni. He wants to make Chizuru suffer because when they were separated, he was taken in by the Nagumo family who treated him harshly for not being a girl. He also hated that Chizuru forgot about him and about her even having a twin brother at all. Because of this, he wants her to feel the pain he felt.

Later on, he admits that he truly loves Chizuru and everything he did was to meet her again. In addition, he asks Chizuru to go with him, because he thinks he is the only one who has the power to protect her. She refuses, since she had promised to the Shinsengumi that she would protect Kondo, though the main reason was because she liked Hijikata. Kaoru decides that if things had come to that, his only option would be to kill her. Right after they start fighting, Chizuru is cornered and saved by Chikage, who kills Kaoru.

Kaoru is the wielder of the long sword that with Chizuru's short sword form a set, both of which are the Yukimura clan's heirlooms.

Chikage Kazama (風間 千景 Kazama Chikage?)
Voiced by: Kenjirō Tsuda (Japanese); Corey Hartzog (English)

Member of the Bakumatsu, enemies of the Shinsengumi. He is the strongest Oni of the West and is after Chizuru, so that they can mate and create stronger Oni. His reasons for this are unknown, but his attempts to take Chizuru have so far been thwarted by the interference of the Shinsengumi. Despite being arrogant and cynical, he is not entirely cold-blooded as he later saves Chizuru (although this might have been for his own purposes) and even escorted her back to the estate rather than harassing her.

Kazama is usually followed by his two demon companions Amagiri and Shiranui, and it is implied by Sen-hime that he is a royal blood, like her. In his Oni form, he grows white horns and his hair fades to match. His eyes also become yellow. But later in the series and OVA he is seen to truly love Chizuru.

Kyuju Amagiri (天霧 九寿 Amagiri Kyūju?)
Voiced by: Ryū Yamaguchi (Japanese); Steven Fenley (English)

Kyūju Amagiri is a very strong red-haired Oni, who prefers to fight with his fists instead of a sword. He's usually seen by Kazama Chikage's side, since he works for him. Strangely, Amagiri seems to dislike fighting needlessly and often tells his opponents not to fight him, in a polite manner. He is rather respectful of his enemies, not even taunting them when in battle. He is also a member of the Satsuma Clan.

In the second season of the anime adaptation, Kazama, Amagiri, and Shiranui unite against Kodo and his army of Rasetsu, which he manages to improve and now can fight efforlessly in broad daylight. Moreover, Amagiri reveals to Saito that, since the source of the Rasetsu's power is not of divine origin unlike the power of true Oni, the superhuman strength, reflexes and regenerative abilities of Rasetsu are fueled from life energy.

Therefore, Rasetsu are simply using in advance energy that was originally meant to be expended over the decades of an ordinary human life, and their lifespans are shortened every time they use their powers.

Kyo Shiranui (不知火 匡 Shiranui Kyō?)
Voiced by: Hiroaki Yoshida (Japanese); Houston Hayes (English)

Kyō Shiranui is a cocky, arrogant Oni who prefers guns to swords. He has long, purple wavy hair which he wears in a ponytail. Initially, he fights alongside Kazama Chikage and Amagiri Kyuuju against the Shinsengumi (and particularly, Harada), in order to abduct Chizuru under Kazama Chikage's orders.

Later on, Harada and Shiranui call it a truce and join forces to defeat Kodo's newly created Rasetsu, after his betrayal. Shiranui serves as a rearguard so that the Shinsengumi can escape the battlefield, opening a path for them. Afterwards, he informs Harada that Kodo is willing to start a war only to get blood to feed his Rasetsu army.

Sen-hime (千姫?)
Voiced by: Ayano Ishikawa (Japanese); Hilary Haag (English)

She is a pretty young girl around Chizuru's age, with long brown hair. She is the princess of oni and protected by her faithful bodyguard, Kimigiku. Sen is first introduced when she protects a child and is threatened by a group of men. Chizuru jumps in, forgetting that she cannot fight very well, and Saito Hajime steps in, knocking the men out with the blunt side of his blade. She later reveals Chizuru's real heritage, even offering twice for Chizuru to come with her and Kimigiku to be better protected.