Topoisomerase IIα has been a representative anti-cancer target for decades thanks to its functional necessity in highly proliferative cancer cells. As type of topoisomerase IIα targeting drugs, topoisomerase II poisons are frequently in clinical usage. However, topoisomerase II poisons result in crucial consequences resulted from mechanistically induced DNA toxicity. For this reason, it is needed to develop catalytic inhibitors of topoisomerase IIα through the alternative mechanism of enzymatic regulation. As a catalytic inhibitor of topoisomerase IIα, AK-I-191 was previously reported for its enzyme inhibitory activity. In this study, we clarified the mechanism of AK-I-191 and conducted various types of spectroscopic and biological evaluations for deeper understanding of its mechanism of action. Conclusively, AK-I-191 represented potent topoisomerase IIα inhibitory activity through binding to minor groove of DNA double helix and showed synergistic effects with tamoxifen in antiproliferative activity.