Abstract:  To explore the effect of cinobufacini on the breast cancer cell line MDA-MB-231. The effects of cinobufacini on intracellular calcium ions and cell membrane potential were studied. Methods: Atomic force microscopy-based technology was used to detect the changes in the morphologic properties of cells at the nanometer scale. The membrane potential of breast cancer cells was determined by staining with fluorescence probe DiBAC4(3) and the calcium ions were stained with fluorescence probe Fluo-3/AM in each group, and then FCM technology  was utilized to measure the changes in fluorescence intensity. Results: The AFM morphologic data showed that the MDA-MB-231 cells in the control group were swollen, but after cinobufacini treatment, the size of cells decreased and the nucleoli became crimpled. Flow cytometry data indicated the cell membrane potential and the calcium ion concentration increased significantly after cinobufacini treatment with different concentrations. Conclusion: Cinobufacini induces the apoptosis of MDA-MB-231 cells, causes the cell membrane potential to depolarize; then, calcium ion channels are opened and calcium ions influx into the cells. Finally, the calcium ion acted as secondary messengers for cell-mediated apoptosis.