Abstract: This study was to investigate the effect of cinobufacin on human gastric cancer MKN-45 apoptosis.After treatment with 0, 60, 70, and 80 μg/L concentrations of cinobufacin for 48 h, the morphology of the cells was observed by laser confocal microscopy. The apoptosis, mitochondrial membrane potential（ ΔΨm）were assayed by flow cytometry. The m RNA and protein levels of Bax, Bcl-2, Cyt C, Caspase 9 and Caspase 3 were measured by RT-qPCR and Western blot. Compared with the control group, the results showed that 0,60, 70, and 80 μg/L concentration of cinobufacin on human gastric cancer MKN-45 cells for 48 h, morphological changes such as cell shrinkage, nuclear lysis, chromatin condensation were observed by laser confocal microscopy, and the percentage of early apoptotic cells and late apoptotic cells increased significantly and the level of ΔΨm decreased. In addtion, the mRNA and protein expressions of Bax, Cyt C, Caspase 9 and Caspase 3 significantly increased（P＜0.01）, while the mRNA and protein expression of Bcl-2 significan-tly decreased（P＜0.01）. The results suggested that cinobufacin induced the apoptosis of human gastric cancer MKN-45 cells through up-regulation of Bax, Cyt C, Caspase 9 and Caspase 3 and down-regula-tion of Bcl-2.