Abstract: Objective: To detect the effects of resibufogenin (RG), a main constituent of Chinese drug Chan Su, on human hepatoma carcinoma Bel-7402 cell line and initial approach the mechanism of its effect. Methods: Bel-7402 was cultured in vitro and then acid phosphatase assay (APA) was used to evaluate the anti-proliferation effect of RG, morphological change was observed by acridine orange (AO) staining, FCM was applied to analyse the apoptotic rate and detect changes in mitochondrial transmembrane potential (△ψm), and the expression of apoptosis-related proteins were determined by western blot. Results: Resibufogenin significantly inhibited the proliferation of Bel-7402 cells, the 50% inhibitory concentration (IC₅₀ at 24,48,72h was 3.8、1.8、1.2 mol/L. 7402 cells treated with RG 10 mol/L) presented the characteristics of apoptotic cell and the apoptotic rate was higher than control groups. RG caused a disruption in △ψm and enhanced the release of CytC then activated Caspase-3. RG inhibited expression of Bcl-2 protein. Conclusion: Resibufogenin could induce the apoptosis of Bel-7402 cells in vitro, which is probably achieved through the mitochondrial-dependent pathway.