Cinobufacini (Huachansu) is a Chinese medicine prepared from the skin of Bufo bufo gargarizans Cantor (Bufonidae), and has long been used in traditional Chinese medicine. In the present study, the anti-retinoblastoma constituent bufalin obtained from Cinobufacini was investigated. Treatment of human retinoblastoma (HXO-RB44) cells with bufalin induced apoptosis which was accompanied by a decrease in mitochondrial membrane potential, activation of caspase-9, caspase-8 and caspase-3, as well as changes in the expression of cytochrome C. Bufalin induced the cleavage of caspase-3 and apoptosis, and it was inhibited by both Z-LETD-FMK and Z-IETD-FMK treatment. Taken together, these results demonstrate that bufalin-induced apoptosis in human retinoblastoma (HXO-RB44) cells involved both intrinsic and extrinsic pathways.