The film is set in 1840s Japan during the final decades of the Tokugawa shogunate. The sadistic younger stepbrother of the current Shogun, Lord Matsudaira Naritsugu of Akashi, sexually assaults and kills at will. Sir Doi Toshitsura (Mikijiro Hira), the Shogun's Justice, realizes the situation will become more dangerous after Naritsugu ascends to a higher political position. After a wronged party of the Mimiya family, publicly commits Harakiri (Harakiri denotes the only way in ancient Japanese way in which slaves demonstrate rejection of their masters. Harakiri means that a slave would rather die by slashing their body rather than continue slaving) as a way of showing disdain for Lord Naritsugu, Sir Doi seeks out a trusted older samurai, Shinzaemon, who served under the former shogun. Sir Doi secretly hires him to assassinate Naritsugu. However, Naritsugu's loyal retainers - led by Hanbei; an old contemporary of Shinzaemon - learn of the plot by spying on Doi's meetings. Naritsugu is so ruthless that he slaughters the entire Mimiya family in cold blood, despite Hanbei pleading with him to leave them alone. But despite the incident, Hanbei is sworn to protect Naritsugu.Shinzaemon gathers 11 more samurai, whom Sir Doi knows can be trusted including Shinzaemon's nephew, Shinroukuro. The 12 plan to ambush Naritsugu on his official journey from Edo back to his lands in Akashi. However just before they leave, Hanbei arrives and warns his old colleague that he will suffer grave consequences if he tries to kill Naritsugu.The group, with the legal authority and financial assistance of Sir Doi, buy the help of town Ochiai in order to create a trap. They also enlist the help of a minor lord whose daughter-in-law was sexually assaulted, and son murdered by Naritsugu. With troops, he blocks the official highway forcing Naritsugu to head for the town and the trap. During the assassin's own journey to the town, they are attacked by Ronin paid off by Hanbei to kill the plotters.The group decides to head through the mountains but end up getting lost. In the process they encounter a hunter named Kiga Koyata who becomes their guide, and they adopt him as the thirteenth assassin. The town is converted into an elaborate maze of booby traps and camouflaged fortifications. However, when Naritsugu and his retinue arrive their numbers have been swelled with additional troops. The 13 assassins were no longer facing 70 men-at-arms now there were 200. A lengthy battle follows with Naritsugu and his guards trapped inside the village and attacked on all sides by arrows, explosives, knives, and swords - with the exception of Koyata who fights with rocks in slings. Against this carnage, the deranged noble, Naritsugu, is excited by the violence claiming it's the most fun he has ever. He tells Hanbei that when he ascends to the Shogun's counsel, he will bring back the wars of the Sengoku period.Slowly the assassins are killed but not before they inflict heavy casualties on the Akashi forces. Eventually Naritsugu and Hanbei are cornered by Shizaemon and Shinrokuro. After Shizaemon kills Hanbei, Naritsugu kicks his loyal retainers head away insulting the older samurai. Contemptuously Naritsugu announces that both the people and samurai have only one purpose and that is to serve their lords. But Shizaemon counters by telling Naritsugu that lords can't live without the support of the people, and that if a lord abuses his powers the people will always rise against them. Naritsugu and Shinzaemon both mortally wound each other. As the lord crawls away in mud crying and experiencing fear and pain for the first time, he thanks Shinzaemon for showing him excitement before Shinzaemon chops his head off.After Shinzaemon dies, Shinrokuro wanders through the carnage. He meets the hunter Koyata who has made a miraculous recovery after being stabbed in the neck by Naritsugu. The pair then make their own ways out of the town. An epilogue states that the Shogun and his government covered up what really occurred announcing that Naritsugu died of illness on the journey back to his lands. In 23 years, the Tokugawa Shogunate would be overthrown with the Meiji Restoration.