Abstract: Objective To study the apoptosis of colon cancer cell line HT-29 cells induced by bufotoxin and its mechanism. Methods Inhibitory effect of bufotoxin on proliferation of colon cancer HT-29 cells was detected by MTT test.Changes in cell morphology were observed with Wright-Giemsa staining.Apoptosis of colon cancer HT-29 cells was analyzed by flow cytometry.Expressions of livin,Bcl-2,Bax and capase-3 protein were detected by Western blotting. Results Bufotoxin inhibited the proliferation of colon cancer HT-29 cells in a time–and dose-dependent manner and induced their apoptosis.Typical apoptotic bodies were observed.Flow cytometry showed a significant peak of hypodiploid apoptosis.The expressions of livin and Bcl-2 were down-regulated while the expression of Bax was up-regulated during the apoptosis of colon cancer HT-29 cells induced by bufotoxin.Bufotoxin also activated caspase-3 protein. Conclusion Bufatoxin induces apoptosis of colon cancer HT-29 cells possibly by down-regulating the expressions of livin and Bcl-2 protein,up-regulating the expression of Bax protein and activating caspase-3.