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The story follows Travis Touchdown, who is a stereotypical otaku – his motel room decorated with professional wrestling and anime collectibles – living in near poverty in the No More Heroes motel of the fictional town of Santa Destroy, California.[10] After winning a beam katana in an internet auction, he runs out of money to buy video games and wrestling videos. After meeting with Sylvia Christel, he accepts a job to kill Helter Skelter, also known as "the Drifter," which earns him rank 11 by the United Assassins Association, a governing body of assassins. Realizing that he has the opportunity to make it to the top, he sets out to secure himself the coveted position of number one assassin in the UAA.

After killing the tenth ranked assassin, Sylvia reveals that if Travis stops killing, he'll eventually be targeted by other aspiring assassins. Travis, now convinced there's no way out, goes on to kill every other assassin except for number eight, Shinobu, whom he spares because he wants her to get stronger. During the 5th ranked battle he meets Henry, a mysterious Irish man who wields a beam katana similar to Travis' own.

Before meeting the top-ranked assassin, Travis is told that the UAA was just an elaborate con set up by Sylvia, who overheard his drunken ramblings and organized his entry so that he could finally have revenge on his half-sister, Jeane, who murdered his parents. Jeane reveals in a fast-forwarded cutscene that their father had sexually abused her all her life, thus forcing her to become a prostitute to survive and become a killer. Travis is eventually saved by Shinobu and kills Jeane.