Abstract: Objective To investigate the effects of telocinobufagin on viability and apoptosis of colorectal cancer (CRC) cells and explore the mechanism of telocinobufagin-induced apoptosis. Methods MTT assay was performed to detect the viability of CRC cells exposed to telocinobufagin. Nuclear staining with Hoechst 33342 and flow cytometry were used to analyze the cell death of CRC cells. Expressions of proteins related with cell apoptosis and oxidative stress were determined with Western blotting. Resuts Telocinobufagin decreased the viability of CRC cells in a time- and dose-dependent manner. The presence of karyopycnosis and apoptotic bodies together with the results of flow cytometry suggested that telocinobufagin induced cell apoptosis to cause cell death. Western blotting showed that telocinobufagin exposure of the cells resulted in upregulated p53 and Bax protein expressions and promoted cleavage of caspase 9 and PARP. Telocinobufagin induced phosphorylation of Bad and PARP cleavage, and suppressed phosphorylation of IΚBα and TAK1 and expression of survivin in the cells. Conclusion Telocinobufagin can decrease the viability of CRC cells by inducing cell apoptosis, which involves p53-mediated Bax activation and inhibition of the IAP pathway.