Violacein is a blue-purple pigment extracted from soil bacteria and has been reported to have anticancer activity. In this study, we assessed the anticancer activity of violacein and confirmed the cytotoxicity and induction of apoptosis in colon cancer cell lines. It was confirmed that violacein-induced apoptosis follows two pathways. In the first pathway, activation of caspase-8 (an inducible caspase) activates caspase-3 (an executive caspase), which induces apoptosis in cancer cells. In the second pathway, cytochrome c is released from mitochondria and activates caspase-9 (an inducible caspase), which in turn activates caspase-3 (an executive caspase), which induces apoptosis in cancer cells. These findings suggest that violacein might be useful as a therapeutic agent for colorectal cancer in the future.