Abstract: Objective To investigate the effect of bufalin on the proliferation and apoptosis of melanoma cells B16, and explore its inhibitory action on melanoma. Methods The effects of bufalin on the proliferation of melanoma cells B16 were detected by MTT. The morphological changes of the cells was observed by fluorescence staining with Hoechst 33342. Cell cycles were detected by flow cytometry. Results Bufalin significantly inhibited the proliferation of melanoma cells B16 in a time- and dose-dependent manner. IC50 were 37.80, 6.00 and 9.12 μmol/L respectively after 24, 48 and 72 h co-culturing with bufalin. Typical morphological characteristics of apoptosis were shown by fluorescence staining after 24 h. Investigation of cell cycles indicated that the S-stage cells were remarkably reduced, and cell cycles were arrested at G0/G1 in a time-dependent manner. Conclusion Bufalin could induce cell apoptosis by arresting cell cycles of melanoma cells B16.