Apoptosis plays a critical role in acetaminophen (AAP)-induced hepatic injury, since inhibiting apoptosis also prevents the development of acute liver failure. In this study, the mechanism of apoptosis induction by AAP was investigated in the human hepatoblastoma cell line HuH7. AAP caused marked cytotoxicity in HuH7 cells as a result of apoptosis. Processing of execution caspases to their corresponding active fragments and cleavage of cytokeratin-18 were observed, supporting a role of caspases in AAP-induced apoptosis. The manifestation of apoptosis was preceded by a translocation of cytochrome c from mitochondria to the cytosol. In conclusion, AAP induces apoptosis in human hepatoblastoma HuH7 cells through mitochondrial cytochrome c release and caspase activation.