Acetaminophen (APAP) overdose is a major cause of acute liver failure. Peripheral 5-hydroxytryptamine (serotonin, 5-HT) is a cytoprotective neurotransmitter which is also involved in the hepatic physiological and pathological process. This study seeks to investigate the mechanisms involved in APAP-induced hepatotoxicity, as well as the role of 5-HT in the liver's response to APAP toxicity. We induced APAP hepatotoxicity in mice either sufficient of serotonin (wild-type mice and TPH1-/- plus 5- Hydroxytryptophan (5-HTP)) or lacking peripheral serotonin (Tph1-/- and wild-type mice plus p-chlorophenylalanine (PCPA)). Mice with sufficient 5-HT exposed to acetaminophen have a significantly lower mortality rate and a better outcome compared with mice deficient of 5-HT. This difference is at least partially attributable to a decreased level of inflammation, oxidative stress and endoplasmic reticulum (ER) stress, Glutathione (GSH) depletion, peroxynitrite formation, hepatocyte apoptosis, elevated hepatocyte proliferation, activation of 5-HT2B receptor, less activated c-Jun NH₂-terminal kinase (JNK) and hypoxia-inducible factor (HIF)-1α in the mice sufficient of 5-HT versus mice deficient of 5-HT. We thus propose a physiological function of serotonin that serotonin could ameliorate APAP-induced liver injury mainly through inhibiting hepatocyte apoptosis ER stress and promoting liver regeneration.