Lindsay (Ashley C. Williams) and Jenny (Ashlynn Yennie) are two American tourists in Germany whom attempt to drive to a local nightclub when their car breaks down in the nearby woods. After refusing a ride from a dangerous looking truck driver, they walk through the woods looking for them when it begins to rain. They stumble upon a rural country house owned by a crazed surgeon named Dr. Heiter (Dieter Laser). Dr. Heiter pretends that he is phoning for them when he serves the two young women drugged water which upon drinking it they pass out. The women awake in a makeshift medical ward in the basement and witness Heiter informing a kidnapped truck driver (Rene de Wit) that he is "not a match" and killing him.
When the women wake up a second time, Heiter has secured a new male captive, Japanese tourist Katsuro (Akihiro Kitamura). The doctor explains to his three captives that he is a world-renowned expert at separating conjoined twins, but dreams of making new creatures that share a single digestive system. He describes in detail how he will surgically connect his three victims mouth-to-anus. After Lindsay fails in an attempt to escape, Heiter performs the surgery on his victims, placing Lindsay in the middle, Katsuro at the front, and Jenny at the rear. Before beginning the operation, Heiter explains to Lindsay that he had experimented with creating a 'three dog', also joined mouth-to-anus, which died shortly after surgery. Heiter tells Lindsay that the middle dog of his creation experienced the most pain, and as a punishment for her escape attempt she will become the middle part of his "human centipede".
Once the operation is complete the doctor tries to train his centipede as a pet, and watches with great delight as Lindsay is forced to swallow Katsuro's excrement. However, Heiter eventually becomes irritated after being kept awake day and night by the constant screaming of his victims and he soon realizes that Jenny is dying from blood poisoning caused by the surgery.
A few days later, two local detectives, Kranz (Andreas Leupold) and Voller (Peter Blankenstein), arrive at the doctor's house to investigate the disappearance of tourists, Heiter decides to add them to his centipede as replacements for Jenny. Heiter fails in an attempt to drug the detectives, and they leave the house to obtain a search warrant.
The victims attempt to escape from the ward, crawling up the stairs, and Katsuro attacks Heiter, stabbing him in his left leg with a scalpel. Their attempt to escape ultimately fails. Katsuro confesses to the doctor, in Japanese, that he deserves his fate because he had treated his family poorly. He then fatally cuts his own throat with a shard of broken glass. Just then, the detectives return to the house and conduct separate searches, as Heiter hides in the basement near his swimming pool. Kranz finds the makeshift ward and then hears a gunshot. He discovers Heiter's victims before finding Voller dead in the swimming pool after being shot. Heiter comes out of hiding and shoots Kranz in the stomach, and Kranz responds by shooting Heiter in the head. Kranz then falls in the pool, dead.
Back in the house, Jenny and Lindsay hold hands as Jenny dies from her blood poisoning. Despite being aware that their captor is dead, Lindsay is left alone in the house, trapped between her two deceased fellow captives.