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This study was performed to investigate the cytotoxic activity from Vitex rotundifolia. V. rotundifolia was extracted with methanol, ethanol, and acetone, and then the cytotoxic effect of these extracts was measured by the MTT reduction assay and morphological assay on the HT-29 human colon carcinoma cells. Among the three extracts, the acetone extract showed the highest cytotoxic activity on the HT-29 cells in a dose-dependent manner with an IC∧50 value of 10 ㎍/ml. The acetone extract was further fractionated sequentially with n-hexane, diethyl ether, ethyl acetate, and water layer according to the degree of polarity.