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Tsukune Aono, an average teenager, is unable to get into any local high schools due to his poor grades. However, his parents are happy to have him enroll in a private school called Yokai Academy, which he discovers is a boarding school for monsters. The school teaches monsters how to coexist with humans, including disguising as them, but any real humans on campus are to be killed. Although he fears for his life, Tsukune befriends a beautiful vampire girl Moka Akashiya, who likes the taste of his blood. Fighting a school bully, he accidentally removes the rosario around Moka's neck, and discovers that she turns into a powerful vampire with a completely different personality.
Because of Moka, Tsukune decides to stay at Yokai, and becomes friends with some formerly antagonistic students, including Kurumu Kurono, a busty succubus and Yukari Sendo, an eleven-year-old witch. They join the school's Newspaper Club and wind up fighting a variety of students and teachers, who try to use their monstrous powers to bully or to control one or more of the members for their own selfish or destructive ends. On a club trip to the human world, they meet witch Ruby Tojo, who tries to defend a field of sunflowers from being torn down by developers, and her mistress Lady Oyakata, who hates all humans. They meet Mizore Shirayuki, a snow fairy student who becomes Tsukune's "stalker". A group of "Monstrels" harass the club, causing Moka to transfuse blood into Tsukune to the point where he turns into a ghoul. Ruby, having recovered from injuries, joins the school's staff as the Headmaster's assistant. An evil group called the Anti-Schoolers threaten to ruin the school's festival and destroy the barrier between the human and monster worlds. When Tsukune's cousin manages to sneak onto the school grounds, the club has a hard time keeping the monster school a secret. When the school is temporary closed because of damage, Tsukune returns home to the human world only to have all four girls follow him and hide in his room (to the shock of his mother).
In Rosario+Vampire: Season II, Koko Shuzen, Moka's younger sister, enrolls; she, Ruby, and Mizore join the Newspaper Club. On an excursion to Mizore's homeland, they learn of a dangerous organization called Fairy Tale, which seeks to destroy the human world, and must fight to keep the coexistence between humanity and monsters intact. Tsukune continues to train; the Newspaper Club visits the human world where they meet San Otonashi, a former club member, as they run into trouble with Fairy Tale's 7th Branch office. A Chinese transfer student, Fangfang Huang tries to recruit Tsukune to join his mafia family. When Moka's inner personality spends a day at school, she is unable to return to the seal, so the Newspaper Club heads to China to get the rosario fixed.
At Hong Kong, Tsukune and the gang learn the truth about Moka's identity, that she was infused with First Ancestor blood by her mother Akasha Bloodriver who sacrifices herself to protect them from Alucard, the original First Ancestor vampire. Moka's eldest sister Aqua infiltrates the Huang manor and captures Moka to bring to Fairy Tale, as she is the key to reviving Alucard. While his friends train their powers, Tsukune has his body altered by Tohofuhai to handle the upcoming challenges.
Tsukune and the gang infiltrate the Hanging Garden, a sky fortress that serves as Fairy Tale's headquarters. They face opposition from their leader Gyokuro Shuzen, who sends the leaders of the Fairy Tale Branches after them for a series of fights. The group eventually reaches Moka, but must face her sisters Aqua and Kalua. Gyokuro snatches Moka's rosario in order to control Alucard. The Masked King reveals himself to be a clone of Alucard's original vampire form. He reveals that he has manipulated both Fairy Tale and its opposition to determine whether the rosario could control him, and then merges with his monster self, who goes on a rampage in Tokyo.
Moka is mortally wounded, but Tsukune revives her by removing all his holy locks and transforming himself into a First Ancestor vampire. They beat down Alucard, but he revives and reveals that he has hatched eggs in Japan's major cities. Tsukune's friends and allies, along with the two other Dark Lords, band together to fight Alucard and his clones. Tsukune puts Moka's rosario on Alucard, which awakens Akasha inside his monster body. Akasha tells Alucard to give up the fight, and they cast a self-disintegration spell. Now that the existence of monsters is known in the human world, coexistence is now even harder to achieve, however, Tsukune is confident that he and his friends can do it.