Objective:To explore the effect of cinobufacini on the production of reactive oxygen species(ROS) and mitochondrial membrane potential in human cancer cell line MDA-MB-231.Method:The MDA-MB-231 cells treated with different concentrations of cinobufacini for 24 h,then used probe rhodamine-123 as mitochondrial membrane potential probe and used DCFH-DA probe as ROS probe,the levels of mitochondrial membrane potential and ROS of MDA-MB-231cells were tested by flow cytomentry.Result:After treated with different concentrations of cinobufacini,the ROS levels were significantly higher than control group and the mitochondrial membrane potential levels were lower than control groups significantly.Conclusion:Cinobufacini could increase the cytosolic ROS levels in MDA-MB-231cells and decrease mitochondrial membrane potential levels induced mitochondrial apoptosis pathway.