The story begins with a battle between the forces of Yoshitatsu of the Saito clan and Nobunaga of the Oda clan in a huge field. Samanosuke is seen overlooking the battlefield on top of a mountain when some Oda soldiers come to take his life. However, the soldiers are no match for Samanosuke's amazing swordsmanship and superior fighting skills. As victory seems imminent, Nobunaga laughs until a stray arrow pierces his throat. Afterwards, Samanosuke receives a letter from Princess Yuki of the Saito clan. In the letter, Yuki says that maids and servants have been disappearing and she suspects monsters to be the culprit. She also request that Samanosuke saves her from the castle before she ends up as one of the victims. Samanosuke and Kaede arrive at the castle too late to rescue Yuki. The player then sees an army of soldiers marching with a pale but living Nobunaga leading them.Samanosuke and Kaede split up and head to the keep. After a little ways, Samanosuke finds two ninja genma and an unconscious Princess Yuki. Samanosuke is unable to defeat them before they escape. As Samanosuke and Yuki have a brief reunion, a gigantic club wielding genma named Osric emerges from the ground. Samanosuke attempts to defend Yuki, but is easily dispatched and sent flying against the wall. As he lies unconscious, he is visited by 12 gods of the Oni clan. They give Samanosuke the power to destroy the genma and absorb their souls with a mystical gauntlet. As he enters a cave, he finds the thunder orb and the Raizan. As he finds the entrance to the keep, he realizes it is sealed and he doesn't have the power to open it. In a dry moat, he finds and kills Osric. As it dies, it destroys a part of the wall which leads to a hallway. In the room at the end, is a laboratory and a genma scientist named Guildenstern operating on a human. Guildenstern reveals that he resurrected Nobunaga and that he made a pact swearing his allegiance to the genma. Guildenstern then escapes and sends a creature called a Reynaldo against Samanosuke. He defeats it and obtains the fire orb and the Enryuu in the laboratory. With the means to enter the keep now, he goes inside to find the entire place a derelict. Within the keep he encounters a man named Tokichiro Kinoshita wrestling with a small boy. After the boy escapes, Tokichiro states that he is a servant of the Oda clan and wonders if Samanosuke would be interested in joining. Samanosuke refuses, saying that he controls his own life. Tokichiro says he will not give up and leaves the room laughing. As he moves on, Samanosuke sees Kaede chasing the young boy. Samanosuke only learns his name is Yumemaru before he escapes again and Kaede goes after him. Kaede later learns that he is an orphan and Yuki has raised him like a brother.

As Samanosuke nears the top of the keep, he sees Yumemaru in the hands of a ninja genma. Before he can give chase, Tokichiro confronts him once again with an offer to join the Oda clan. He also reveals that something called the dark ceremony will take place soon. In it, Princess Yuki's skull will be filled with her blood. Fortinbras, the demon king, will bless it and Nobunaga will drink it in order to become powerful and destroy the Saito clan; while he offers human sacrifices to the demons. When he finds the room Yumemaru is in, he realizes he doesn't have the power to unseal the magical barrier. He fights a strong genma named Marcellus on the roof and receives the wind orb and the Shippuu when it is defeated. With this, he is able to rescue Yumemaru and brings him to a secure room in the keep. As Samanosuke tells him about the world, a maid comes in saying that Princess Yuki is underground. Samanosuke tells Kaede to stay with Yumemaru and the woman while he looks for Yuki. While he searches, he finds himself once again in the presence of Tokichiro. With one last request to join the Oda clan, he activates a device and Samanosuke is pulled down into the earth. Meanwhile, Kaede is rendered unconscious by a genma which looks like Samanosuke and Yumemaru is kidnapped by a woman. When she awakens, Kaede is led to the prison and finds Yuki locked in a cell. Before she can free her, Guildenstern arrives from the darkness of the cell and leaves Kaede to die at the hand of a powerful genma. She escapes once it is dead.

Samanosuke awakens and kills his counterpart in the underground passage. He makes his way back into the keep and arrives in the room where the woman and Yumemaru are. The woman reveals her true form which is that of an insect-like Genma called Hecuba and erects a door to the demon world which she flies into with Yumemaru. When Samanosuke faces her, she reveals that Yumemaru is to be killed in front of Yuki which will heighten her sorrow, thus granting Nobunaga even more power when he uses Yuki's skull to drink her blood. Samanosuke and Kaede eventually vanquish the insect Genma. As Samanosuke makes his way through the demon door, he encounters Guildenstern who summons a more powerful version of Marcellus who now has a red shading all over his body since he didn’t have the wind orb like his previous incarnation. After defeating him, he makes his way into Fortinbras' throne room. There he finds Yuki and Yumemaru trapped on the upper level. Before Samanosuke can free them, Fortinbras enters the room. After a brief conversation, the two clash. It would seem that Samanosuke killed Fortinbras and he frees Yumemaru and Yuki as Kaede enters the room.

As they exit, the place comes crashing down and Fortinbras finds the strength to grab Samanosuke. A Kaede, Yumemaru, and Yuki escapes as the strain of Fortinbras's grip causes Samanosuke to spit up blood. Some of the blood falls on the gauntlet and Samanosuke becomes an Onimusha and kills Fortinbras by stabbing him in his central eye. As Samanosuke transforms back into a human, he sees Nobunaga's reflection in Fortinbras' eye. The two stare at each other as the room continues to collapse. During the ending sequence Yuki and Yumemaru follow Samanosuke’s advice and travel the world. After the end credits, Samanosuke is seen alive, viewing Inabayama Castle from afar, still possessing the gauntlet. Kaede spends 12 years looking for Samanosuke until she finally finds him (as seen In Onimusha Blade Warriors).

Historical background Samanosuke is the nephew of Mitsuhide Akechi, the man historically credited to have actually killed Nobunaga Oda over 20 years later. It is rumored that Samanosuke is an alternative name for one of Mitsuhide's actual nephews and may have been born in 1536 or 1538. It is also rumored that he went by the name Hidemitsu Akechi, which is used in Onimusha 3. Some speculate that he is a nephew or cousin to Mitsuhide, others say brother-in-law, but not much is said about Samanosuke (Hidemitsu), just that he helped Mitsuhide in the attack on Nobunaga, and lived somewhat longer than Mitsuhide. His death is also a mystery; it was either seppuku or death in battle against Hideyoshi's troops. In the game, Nobunaga is "killed", to be later resurrected by demons, in the Battle of Okehazama. Historically, he was actually mistakenly reported to have been felled by an arrow in the Okehazama battle.