The film concerns a deranged German surgeon who kidnaps three tourists and conjoins them surgically, mouth to anus, forming the eponymous "human centipede".

According to Six, the concept arose from a joke he had made with friends about punishing a child molester by stitching his mouth to the anus of a "fat truck driver".

Lindsay and Jenny, tourists from New York visiting Germany, get a flat tire on their way to a night club and seek help at the house of misanthropic, psychopathic surgeon Dr. Josef Heiter.

After Lindsay tries to escape and fails, Heiter decides to make her the middle part of the centipede, the most painful position for the healing process, as punishment.

Heiter becomes irritated after being kept awake by the screaming of a caged Katsuro (who, as the front part of the centipede, has his mouth free and is still able to speak, but in Japanese) and by the constant moaning of the women, threatening to remove their vocal cords.

Katsuro confesses to Heiter that he deserves his fate because he treated his family poorly, then takes his own life by slitting his throat with a glass shard.

Voller begins to feel ill from the earlier drugging and Heiter stabs him with a scalpel that he had pulled from his foot during Katsuro's attack, killing him.

The inspiration for the film's plot came from a joke that writer/director Tom Six once made to his friends about punishing a child molester they saw on TV by stitching his mouth to the anus of an overweight truck driver.

"[20] A major influence for The Human Centipede was Pier Paolo Pasolini's controversial 1975 Italian drama film Salò, or the 120 Days of Sodom, which was notable for its scenes depicting intensely graphic violence, sadism, and sexual depravity, as was the work of Japanese director Takashi Miike.

[22] Further inspiration came from Six's previous role as a director on the Dutch series of Big Brother, where he had been able to observe people who "did crazy things when they were alone and thought they were not (being) watched.

[12] Six did this in an attempt to lull audiences into thinking they are watching a conventional horror film, therefore making Dr Heiter's treatment of his victims more shocking.

[41] Laser remained in character as Heiter throughout the filming process, often shouting at the rest of the cast on set,[16] and wherever possible staying away from the other actors and crew between scenes to preserve a level of separation.

[49] Due to the discomfort of spending long periods on their hands and knees, the actors playing the centipede were given massages at the end of each day of filming.

[51] The Human Centipede contains relatively few gory images; little of the surgical procedure is depicted directly,[53] no excrement is shown on screen,[54] and according to Kim Newman in Empire, it is "never quite as outrageous as it threatens to be.

Roger Ebert, writing in the Chicago Sun-Times, stated that he felt the film had been "deliberately intended to inspire incredulity, nausea and hopefully outrage.

"[20] However, writing in The Guardian, David Cox noted that he had been unable to trace the source of this quote as the "most horrific film ever made" and had contacted Six to attempt to ascertain the origin of the judgement.

When asked by Cox as to what Six regarded as the "most horrific" film, Six stated he in fact believed it to be Pier Paolo Pasolini's Salò, or the 120 Days of Sodom.

[82] Giving the film three stars out of five, Empire writer Kim Newman stated that "underneath an extremely repulsive concept, this is a relatively conventional horror movie.

"[55] Variety Magazine writer Peter DeBurge criticised the film's lack of social commentary, stating that it could not "be bothered to expand upon its unpleasant premise, inviting audiences to revel in its sick humor by favoring Dr. Heiter ... and characterizing the victims as shallow expendables.

"[83] Writing in Entertainment Weekly, Clark Collis was broadly positive about Dieter Laser's performance as the Doctor, and praised Six's direction, saying Six "has put together his nightmare yarn with Cronenbergian care and precision.

"[85] Sukhdev Sandhu of The Daily Telegraph was generally negative about the film, stating, "The Human Centipede has its moments, but they're largely obscured by umpteen holes in the plot as well as by reams of exposition,"[86] and that it was "an ultimately underwhelming affair that's neither sick or [ ⁠sic ⁠] repellent enough to garner the cult status it so craves.

"[86] The New York Times review by Jeannette Catsoulis noted that whether the film was "a commentary on Nazi atrocities or a literal expression of filmmaking politics, the grotesque fusion at least silences the female leads, both of whose voices could strip paint.

Martin compared Six's claims to those of Kroger Babb and William Castle, who had also made "grand promises" about what they were putting on screen, in a bid to lure audiences.

[94] The plot of Full Sequence involved a centipede made from twelve people, featured a largely British cast, and was given the tag-line "100% medically inaccurate".

[108] The South Park episode "HUMANCENTiPAD" saw character Kyle Broflovski unwittingly agreeing to become a part of a "Human CENTiPAD" after failing to read the full details of an Apple user license agreement.

[109] The website Funny or Die featured a sketch where the freed victims of a human centipede, now separated, but scarred physically and mentally, argue at a survivors' meeting.

[111] "Red Flags", a song by Tom Cardy and featuring Montaigne, is about a man who is first repulsed by, and then swept up in his dates obsession with the film.

[112] In January 2016, Tom Six revealed on Twitter that production of a graphic novel adaptation of The Human Centipede was underway, along with posting an image of a test printed copy.

[118][119] The story of the graphic novel will feature the events of the first film but it will display things that occurred before, and an epilogue that will shed light on the fate of the character Lindsay.

[120] Although Tom Six openly stated he viewed The Human Centipede as a trilogy, he told Bloody Disgusting in an interview that if he had to make a fourth installment he did have some ideas.