In the present study, we investigated the antiproliferative activity of Naja ashei full venom (NAV) on human colorectal cancer cells. The NAV-induced antiproliferative effect was associated with cell cycle arrest in S phase and increased number of cells with sub G0/G1 DNA content, which is considered a marker of apoptosis. Apoptosis has also been confirmed with annexin V/PI staining. Furthermore, flow cytometric analysis revealed loss of mitochondrial membrane potential with concomitant increase in cytochrome c and Smac/DIABLO protein content. These effects were associated with the activation of caspase-9 and caspase-3, as well as with PARP cleavage. Moreover, phosphorylation of antiapoptotic Bcl-2 protein in NAV-treated HCT116 was observed. In conclusion, our study for the first time documented antiproliferative/pro-apoptotic effect of NAV in colorectal cancer cells. Our results strongly suggest the involvement of mitochondria in NAV induced apoptosis of cancer cells. Future studies are needed to further examine the potential of NAV in the treatment of colon cancer.