The proliferation of a human colon carcinoma cell line, COLO 201, was effectively suppressed through apoptosis in the presence of flavonoids, an ethanol extract from Vitex agnus-castus fruits. The induction of apoptosis was not inhibited by the presence of an anti-oxidant, N-acetyl-L-cysteine, whereas only HO-1 gene expression levels increased among other typical oxidative stress-associated genes examined after Vitex treatment. These results suggest that Vitex treatment activates a pathway associated with HO-1 gene activation, resulting in the induction of apoptosis in COLO 201. Results also implicate a potential clinical chemotherapeutic application of Vitex for the treatment of colon cancer patients.