Evodiamine, an alkaloid isolated from the Chinese herbal plant Evodia rutaecarpa, was found to be a dual inhibitor of both human topoisomerase I and II enzymes. Testing showed evodiamine has potent cytotoxicity against human leukemia cell lines with IC50 values of 34.35 μM and 90.87 μM. Further experiments revealed evodiamine inhibits the catalytic activities of topoisomerase I and II, but does not induce DNA strand breaks or change the cell cycle distribution, suggesting its anticancer activity is through inhibition of topoisomerase enzymatic function rather than DNA damage.