Asuka, Ikaruga, Katsuragi, Yagyu and Hibari are five school girls who secretly train in the ninja arts. As they are sent by their mentor, Kiriya, to handle some delinquents in town, Asuka meets a girl named Homura and helps guide her through town, unaware that she is secretly an enemy shinobi. Meanwhile, the others encounter the delinquents, who are soon revealed to be wooden puppets controlled by an enemy shinobi. As the girls manage to beat the puppets, Asuka remains blissfully unaware of Homura's true alliance.

As the girls train in summoning animal spirits, Asuka is the only one having trouble summoning hers. Her grandfather, the legendary shinobi Hanzou, comes to visit and informs her that their family's spirit is the tree frog, which Asuka is afraid of. As Asuka attempts to get used to frogs, Hanzou informs Kiriya that the evil Serpent Girl's Academy is behind the puppet attack. On their way home, Asuka and Ikaruga are attacked by one of the enemy shinobi.

The dark shinobi engages Ikaruga in battle and overwhelms her with her array of weapons before taking her leave. Later, Ikaruga is informed that her brother, Murasame, who she replaced to become the shinobi successor of her family, has gone missing. Later that night, Ikaruga encounters Murasame in her room stealing her sword, Hien, but she manages to send him packing easily enough. After being ejected from the household, Murasame is met by Hanzou, who reminds him that being a shinobi isn't the only way of life. After receiving some advice from Hanzou, Ikaruga decides to start giving Asuka summoning lessons, managing to help her get over her fear of frogs and learn to use her summoning art.

Kiriya takes the girls to Shinobi Island to train. Two more dark shinobis, Mirai and Hikage, attacked. As with the other battles, neither Mirai nor Hikage took their battles seriously and left rather abruptly. However, Mirai revealed to Hibari that she is a freshman at the Serpent Academy for Girls, leading Kiriya to inform his students that these dark shinobis are also from that same academy.

The Hanzo girls train even harder to be ready for the Hebijo shinobi's next move. The Hebijo girls sneak onto campus to locate the Hanzo girls' training facility. Upon finding the old schoolhouse, it is placed in a kekkai, trapping the Hanzo girls inside!

The Hanzo shinobi continue to fight one-on-one against the Hebijo shinobi. When Yagyu manages to defeat her opponent and free herself from her pocket of kekkai space, she attempts to enter Hibari's pocket to protect her, at great physical risk to herself.

Still reeling from the blow they were dealt by Hebijo, Kiriya decides to ramp up the girls' training with a mountain-climbing training excursion. This so-called "Hai-kin-gu" training provides many challenges to the Hanzo team, both together and separately.

Kiriya's training of the girls turns especially strict. Afterward, he reminisces about the time two years earlier when Ikaruga and Katsuragi were new students. As Asuka, Hibari, and Yagyu join in later on, the road to the team's formation is revealed.

Under Haruka's control, Hibari breaks into Hanzo Academy's depository and takes the super-secret ninja art scroll. After Haruka leaves with the scroll, Hibari feels that she has betrayed the others, and runs away from the academy to get the scroll back.

With the map from Suzune, the Hanzo Academy shinobi prepare to attack the Hebijo base. As Hibari learns more about Hebijo's training methods, she is determined to take back the secret scroll. Suzune gets in hot water for luring the Hanzo shinobi to Hebijo.

Yomi waits in ambush to keep the Hanzo shinobi from moving forward. As Ikaruga takes matters into her own hands, Hibari is desperate to escape her cell and tell the others not to fight the Hebijo students, as it will mean their deaths if they lose.

Fierce battles continue to be waged as the Hanzo Academy shinobi try to reach the castle's top floor. Meanwhile, outside, Kiriya, Daidoji, and Suzune have a confrontation of their own. With everything at stake, Asuka and Homura engage in one last showdown.