Background: ONTD, a novel synthetic triterpenoid from enoxolone, is a promising anticancer agent that has shown strong activity against a wide variety cancer types in vitro.Methods: The cytotoxic activity was evaluated by MTT assay.Flow cytometry was employed to detect mitochondrial membrane potential, apoptosis and ROS.Western blot was used to analyze signaling pathways.Results: ONTD concentration-dependently reduces Bel-7402 cells without affecting normal human liver cell line L-02 cells.ONTD-induced apoptosis of was characterized by the generation of intracellular reactive oxygen species (ROS), loss ofmitochondrial membrane potential (△Ψm), the release of apoptotic inducing factor (AlF) and cytochrome c (Cyt c) from mitochondria and cleavage of PARP-1 and procaspases-3,-9.In addition, ONTD activated the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK but not ERK1/2.Treatment with SP600125 (an inhibitor of JNK) and SB203580 (an inhibitor of p38) prior to ONTD markedly rescued Bel-7402 cells from ONTD-induced apoptosis.Conclusions and general significance: ONTD inhibits the growth of Bel-7402 cells through a mitochondrialmediated pathway and triggering apoptosis accompanied with the depletion of intracellular GSH.These data support development of ONTD as a potential agent for hepatocellular carcinoma therapy.