The assassin Helter-Skelter's name is a reference to the Beatles' song, "Helter Skelter".

In the original Wii version, the river overpass in Santa Destroy has a wall underneath it with poor-enough collision detection that if you walk into it in a certain way, Travis will clip through the wall and can walk infinitely directly underneath Santa Destroy, even out of the city. This glitch was fixed in all future re-releases of the game.

The mini-fridge in Travis' room at Motel "NO MORE HEROES" can be used to heal him, even though it's impossible to lose health outside of ranking matches and assassination gigs and Travis' health is always restored after completing or failing a gig. Both the game and its instruction manual still point out this feature regardless. This suggests that there were either plans for enemies to appear in the overworld of Santa Destroy that could harm Travis outside of his missions, or it was implemented purely for creative effect.

The suggestive motion that Travis (and the player) must perform to recharge the Beam Katana resembles a masturbatory act because the japanese word, "自家発電", means both "Manual power generation" and "masturbation".

Travis' bike "Schpeltiger" resembles Kaneda's motorcycle from "Akira."

The assassin Dark Star resembles the Darkstar Skateboard Logo.

Death Metal's saber, the Orange II, was designed by the fictitious "Orange Computers", a parody of Apple Inc.

In the cutscene before the fight with Harvey Moiseiwitsch Volodarskii, the subtitles misspell Volodarskii as "Volodarsky".

Shinobu's outfit in No More Heroes is based off the Japanese "kogal" style.

Trading cards which feature concept art of Letz Shake reveal that early in the development of No More Heroes, he was originally called Let's Shake, and had hair colored red instead of green. He also originally had a Virtual Boy strapped to his face, with the controller attached to his arm.

Shinobu's name is a pun on the word shinobi which means "to steal away". It also means ninja. However "Shinobu" can as a given name mean "endure" or "recall". This depends on how it is written in kanji.

Upon the game's release, executive producer, Yasuhiro Wada, and creator, Goichi Suda, went to a store in Japan to give out No More Heroes based toilet paper and sign copies of the game to celebrate. Unfortunately, not many people came, and they left after 20 minutes. This could be due to them arriving in the middle of a work day unannounced.

The Lovikov Balls are a reference to the Dragon Balls from the anime/manga Dragon Ball. The manual refers to them as being part of a wish-granting dragon - another reference to the Dragon Ball series.

The "To Be Continued" logo at the end of the game's credits is a reference to the Back to the Future film series.

At the end of No More Heroes, Jeane reveals her backstory is allegedly "too terrible" and will "jack up the age rating of the game". She also makes a reference that "you don't want this game to become No More Heroes Forever, a joke about Duke Nukem Forever's long development period. She then proceeds to tell her backstory at a sped up speed so that it is absolutely impossible to understand what she says. Slowing this down reveals a very horrible story:

"You are my half brother. You know that manga called Miyuki? The Japanese one. Well, it's like that. Your father abandoned my mother to run off with your mother. It broke her heart beyond repair. She killed herself. Knowing I had nowhere to go, he took advantage of me. Ever since I can remember, he molested me. We lived in this rotten apartment... And I was his slave. Every day I cursed his soul. I swore that I would kill him one day. But cursing didn't change anything. That's when I decided to become a killer. With no money to pay for training, I paid with my body. But to you, I bet that son of a bitch looked like a hard-working family man. That was all an act. He didn't give two shits about cleaning up my life so I decided to clean it up myself. That's why I do what I do. And I got what I wanted. Killing him in front of your very eyes. That's my story."

The game shares its title with that of an album by The Stranglers. A poster stating "Whatever happened to the heroes?" appears around Santa Destroy, which is a lyric from the song No More Heroes.

The name for Shinobu's katana, the Three Girl Rhumba's Sword, is a reference to the Wire song "Three Girl Rhumba".

Destroyman's alter ego, John Harnet, is a reference to American heavyweight mixed martial arts fighter Josh Barnett, who he bares a strong resemblance to. This was confirmed in a GameTrailers video interview with Suda51.

Silvia Christel's name is an allusion to European actress Sylvia Kristel.

The name for Thunder Ryu's beam katana, the primitive D.O.S., is a reference to the family of operating systems for IBM compatible PCs marketed between 1981 and 1995.