Director Tom Six showed a drawing of the human centipede to all the actresses who showed up to the casting sessions. Many actresses walked out in disgust after discovering what the role actually entailed.
When the Toronto Underground Cinema showed this film, there was a "centipede deal" where if two tickets were purchased, the third one would be free.
When approaching investors prior to filming, the filmmakers told them they wanted to make an international horror film that involved stitching people together but did not mention how exactly they would be stitched, fearing the mouth-to-anus aspect would put off the investors.
The paintings of conjoined twins displayed throughout Heiter's villa were painted by director Tom Six, which he felt contributed to the atmosphere in the house.
The concept of the film arose from a joke director Tom Six made with friends about punishing a child molester by stitching his "mouth to the ass of a very fat truck driver."