Cinobufacini (Huachansu), an aqueous extract from the skins of Bufo bufo gargarizans Cantor, is a well-known traditional Chinese medicine widely used in clinical cancer therapy in China. However, the precise mechanisms induced by cinobufacini in human hepatocellular carcinoma (HCC) cells are still not very clear. The aim of present study was to investigate possible apoptotic mechanisms induced by cinobufacini in HCC cell lines HepG(2) and Bel-7402. We found that cinobufacini treatment resulted in a significant decrease in cell proliferation and induced apoptotic cell death with the increase of treatment time. It indicated that cinobufacini-induced apoptosis was associated with mitochondria-mediated pathway including the loss of mitochondrial membrane potential (Δψm), the increase of Bax/Bcl-2 ratio, cytochrome c release, caspase-9 and caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) degradation. Additionally, cinobufacini also activated Fas-mediated apoptosis pathway obviously as evident by an increase in Fas expression, and caspase-8 and caspase-10 activation. Moreover, the BH3-only protein Bid was cleaved into a truncated Bid (tBid) after cinobufacini treatment. Taken together, these data suggested cinobufacini could induce apoptosis of HCC cells through mitochondria- and Fas-mediated caspase-dependent pathways with the increase of treatment time, which might provide an experimental evidence for cinobufacini treatment of HCC.