The film is set in 1840s Japan, during the waning years of the Tokugawa shogunate. It follows the sadistic younger stepbrother of the reigning Shogun, Lord Matsudaira Naritsugu of Akashi, who indulges in his propensity for violence and sexual assault without consequence. Recognizing the increasing danger posed by Naritsugu's ambition, Sir Doi Toshitsura (Mikijiro Hira), the Shogun's Justice, grows alarmed at the prospect of Naritsugu attaining greater power.
The plot thickens when a member of the Mimiya family openly commits harakiri, a ritual act of defiance against their cruel overlord, Naritsugu. In the aftermath, Sir Doi seeks the expertise of Shinzaemon, a seasoned samurai loyal to the former Shogun, and covertly hires him to carry out the assassination of Naritsugu. However, the plot is soon uncovered by Naritsugu's loyal retainer Hanbei, an old friend of Shinzaemon's, who monitors Doi's clandestine meetings.
In a brutal display of his merciless nature, Naritsugu exterminates the entire Mimiya family, ignoring Hanbei's pleas for mercy. Yet, Hanbei remains bound to his duty of protecting his lord. Shinzaemon gathers a team of eleven other samurai, including his nephew, Shinroukuro, to ambush Naritsugu during his return trip from Edo to Akashi. Just before their departure, Hanbei delivers a chilling warning to Shinzaemon, threatening dire consequences should he attempt to carry out the assassination.
With Sir Doi's support, the group takes decisive measures by recruiting town Ochiai, creating an intricate trap to ensnare Naritsugu. They also gain the backing of a minor lord who bears his own grief; his daughter-in-law was assaulted and his son murdered at Naritsugu's hands. Together, they block the highway to force Naritsugu into the town where their trap awaits. However, unforeseen challenges arise when the assassination group is ambushed by ronin hired by Hanbei to eliminate the schemers.
Opting for a mountainous route, the group finds themselves lost but soon meets Kiga Koyata, a hunter who becomes their guide, ultimately joining them as the thirteenth assassin. They transform the town into a complex maze filled with booby traps and camouflaged fortifications. Yet, when Naritsugu arrives, he is accompanied by an overwhelming force—200 troops instead of the anticipated 70.
What ensues is an intense and chaotic battle, with the assassins launching a blend of arrows, explosives, and blades against Naritsugu and his men. Amidst the frenzy, the deranged lord revels in the violence, declaring that this is the most enjoyable moment of his life. He tells Hanbei of his intent to revive the wars reminiscent of the Sengoku period when he rises to the Shogun's council.
Despite the assassins suffering heavy losses, they manage to inflict significant damage on Naritsugu's forces. Eventually, the final confrontation sees Shinzaemon and Shinroukuro cornering Naritsugu and Hanbei. In an act of defiance, Shinzaemon slays Hanbei, only to be insulted by Naritsugu, who disdainfully kicks the severed head of his loyal retainer.
In a tense exchange, Naritsugu arrogantly proclaims the purpose of people and samurai is to serve their lords. Yet, Shinzaemon counters that lords depend on the people's support and warns that abuse of power will lead to rebellion. In a fatal duel, both warriors deliver mortal wounds to each other. As Naritsugu crawls away, experiencing fear for the first time, he begrudgingly thanks Shinzaemon for the thrill before the latter ends his life.
Following Shinzaemon's death, Shinroukuro wanders through the aftermath, eventually encountering a miraculously recovered Koyata. Together, they make their escape from the town. The epilogue reveals that the Shogun's government concealed the truth of these events, fabricating a narrative that Naritsugu died of illness on his way back. Ultimately, just 23 years later, the Tokugawa Shogunate would be dismantled during the Meiji Restoration.
