OBJECTIVE: To investigate the mechanisms by which allyl isothiocyanate (AITC) exerts its anticancer effects, the present study investigated the cytotoxic effects of AITC and its metabolites on hepatocellular carcinoma HepG2 cells. METHODS: The AITC metabolites, S-(N-allylthiocarbamoyl)-L-glutathione (AITC-GSH), N-acetyl-S-(N-allylthiocarbamoyl)-L-cysteine (NAC-AITC), S-(N-allylthiocarbamoyl)-L-cysteinylglycine (AITC-Cys-Gly), and S-(N-allylthiocarbamoyl)-L-cysteine (AITC-Cys) were synthesized. HepG2 cells were treated with these compounds and AITC and subjected to a cell cycle analysis, HPLC analysis for intracellular AITC metabolites, and intracellular reactive oxygen species (ROS) analysis. RESULTS: AITC, AITC-GSH, and NAC-AITC significantly induced cell cycle arrest in the G CONCLUSION: The conjugation of intracellular GSH with AITC decreased free reduced GSH levels and increased intracellular ROS levels in HepG2 cells, resulting in cytotoxicity, including cell cycle arrest and apoptosis.