Daurinol, a lignan from the ethnopharmacological plant Haplophyllum dauricum, was recently reported to be a novel topoisomerase II inhibitor and an alternative to the clinical anticancer agent etoposide based on a colorectal cancer model. In the present study, we elucidated the detailed biochemical mechanism underlying the inhibition of human topoisomerase IIα by daurinol based on a molecular docking study and in vitro biochemical experiments. The computational simulation predicted that daurinol binds to the ATP-binding pocket of topoisomerase IIα. In a biochemical assay, daurinol (10-100 µM) inhibited the catalytic activity of topo-isomerase IIα in an ATP concentration-dependent manner and suppressed the ATP hydrolysis activity of the enzyme. However, daurinol did not inhibit topoisomerase I activity, most likely because topoisomerase I does not contain an ATP-binding domain. We also evaluated the anti-proliferative activity of daurinol in ovarian, small cell lung and testicular cancer cells, common target cancers treated with etoposide. Daurinol potently inhibited SNU-840 human ovarian cancer cell proliferation through cell cycle arrest in S phase, while etoposide induced G2/M phase arrest. Daurinol induced the increased expression of cyclin E, cyclin A and E2F-1, which are important proteins regulating S phase initiation and progression. Daurinol did not induce abnormal cell and nuclear enlargement in SNU-840 cells, in contrast to etoposide. Based on these data, we suggest that daurinol is a potential anticancer drug candidate for the treatment of human ovarian cancer with few side effects.