Abstract: To investigate the effect of cinobufacin on cell apoptosis of human gastric cancer MGC-803 cells, and explore its molecular mechanisms, the human gastric cancer MGC-803 cells was treated with different concentrations of cinobufacin for 48 h. The changes of cell morphology were observed under the laser scanning confocal microscope, the rate of apoptosis and mitochondrial membrane potential were detected by the flow cytometry, the expressions of Bax, Bcl-2, Cyt-c, Caspase 9 and Caspase 3 gene m RNA and proteins were tested by RT-qPCR and Western Blot.The results showed that after 48 h of human gastric cancer MGC-803 cells treated with cinobufacin, the cells showed obvious apoptosis morphology with the increase of drug concentration, compared with the control group, the mitochondrial membrane potential is significantly reduced, and the early apoptosis and late apoptosis percentage increased. The mRNA and protein expressions of Bax, Cyt-c, Caspase 9, Caspase 3 significantly increased（P＜0.05）, but the mRNA and protein expression of Bcl-2 significantly decreased（P＜0.05）. Therefore, in this study, it was found that in a certain concentration range, cinobufacin could up-regulate Bax, Cyt-c, Caspase 9, Caspase 3 gene expression and down-regulate Bcl-2 gene to induce apoptosis of human gastric cancer MGC-803 cells.