Azumi, one of the last surviving members of a young assassin squad, continues her mission to prevent a civil war in feudal Japan. Alongside her sole remaining comrade Nagara, she is hunted by ninjas hired by General Kanbei Inoue, who seeks revenge for the assassination of his master, Kiyomasa Kato. After a brief refuge in a temple where Nagara heals his wounds, Azumi encounters a bandit named Ginkaku. He is the leader of a rogue group and bears a striking resemblance to Nachi, a fellow trainee and close friend Azumi was forced to kill during her final test.

The assassins originally began their work on the orders of the priest Tenkai, who seeks to eliminate the remaining warlords of the Toyotomi clan who could destabilize the country. Their primary target is Masayuki Sanada, a powerful warlord protected by both an army and a dangerous clan of elite ninja known as the Uenokagashu. To aid them, Tenkai assigns a female government ninja named Kozue to accompany Azumi and Nagara.

As they travel, Azumi is torn by her unexpected attachment to Ginkaku, who reminds her of the peaceful life she might have had. Nagara, convinced by Kozue that Azumi no longer wishes to continue as an assassin, decides they should separate so she can pursue happiness with the bandit. However, Kozue soon reveals herself to be a spy for the Uenokagashu clan, mortally wounding Nagara when they are alone. Azumi discovers the betrayal and kills Kozue, vowing to complete the mission alone.

Determined to stop Sanada, Azumi fights her way through his forces. She battles Tsuchigumo, a hulking enemy ninja who fights with razor-sharp poisoned wires, and manages to turn his own weapon against him. Though victorious, Azumi is cut by the wires and falls into a paralytic stupor. Sanadas concubine and the true leader of the Uenokagashu, Kunio, moves to deliver the final blow. Ginkaku, who had been following Azumi, intervenes and takes the fatal thrust, mortally wounding Kunio but sacrificing himself in the process.

Despite her injuries and grief, Azumi reaches Sanadas camp. She challenges the warlord to a duel, agreeing that regardless of the outcome, his forces will cease fighting the Tokugawa shogunate. Azumi slays Sanada in combat. With her mission complete, she ignores a request to know where she is going and silently departs the battlefield, walking toward an uncertain future alone.