Abstract: Objective To explore the apoptotic mechanism of human hepatic carcinoma cell line HepG2 induced by arsenic trioxide (As2O3). Methods The human hepatoma cell line HepG2 was treated with 0, 2.5, 5 and 10 μmol/L arsenic trioxide for 24 h. Cytotoxicity was tested by MTT assay (additional 25 and 50 μmol/L arsenic trioxide treatment groups), cellular apoptosis were detected by flow cytometry, reactive oxygen species (ROS) level were quantified by DCFH-DA fluorescent probe staining and glutathione content were measured by DTNB method with commercial kits. Western blot assay was used to detect the protein expression of γ-glutamylcysteine synthetase (γ-GCS, GCLC and GCLM subunits) and nuclear factor erythroid 2-related factor 2 (Nrf2). Results With the increase of arsenic trioxide concentration, cellular survival, glutathione content and γ-GCS (GCLC and GCLM subunits) protein expression level decreased (P<0.05); while cellular apoptotic rate, reactive oxygen species level and Nrf2 protein expression increased (P<0.05). Conclusion Arsenic trioxide induces the apoptosis of human hepatoma cell line HepG2 through ROS induction, γ-GCS expression inhibition and cellular glutathione content depletion.