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Objective: The aim of our study was to investigate the effect of Cinobufacini injection on the proliferation and apoptosis of human hepatocarcinoma HepG-2 cells. Methods: Cells proliferation was assessed by MTT assay, cells morphologic was observed by the inverted microscopy, Annexin V/PI stein was used to detect the apoptosis and necrosis of the tumor cells. The expression of TOPOⅠ mRNA and TOPO Ⅱ mRNAwere examined by RT-PCR. Results: Cinobufacini injection significantly inhibited HepG-2 cells proliferation in dose- and time-dependent ways. After Cinobufacini injection intervention,HepG-2 cells showed typical morphological changes: cells changed from polygon into round, chromatin looseness and karyolysis were observed. The percentages of apoptosis were 88.49%, 76.02%, 61.73% corresponding to the 48 h interference of 0.42 μg/mL, 0.21 μg/mL, 0.105 μg/mL Cinobufacini injection, perspectively. RT-PCR assay showed that Cinobufacini injection down-regulated TOPOⅠ and TOPO Ⅱ expression at mRNA level. Conclusion: Cinobufacini can inhibit human hepatocarcinoma HepG-2 cells growth and induce tumor cells apoptosis, the mechanism of which might partly relate to the down-regulation of TOPOⅠ mRNA and TOPO Ⅱ mRNA induced by Cinobufacini injection.