Ethonafide is an anthracene-containing derivative of amonafide that belongs to the azonafide series of anticancer agents. The lack of cross-resistance in MDR-expressing cancer cell lines and the absence of a quinone and hydroquinone moiety make ethonafide a possible less-cardiotoxic replacement for existing anthracene-containing anticancer agents, such as mitoxantrone and doxorubicin. For this study, we investigated the anticancer activity and mechanism of action of ethonafide in human prostate cancer cell lines. Ethonafide was cytotoxic against three human prostate cancer cell lines at nanomolar concentrations. Ethonafide was also found to be better tolerated and more effective at inhibiting tumor growth compared to mitoxantrone in DU 145 prostate cancer-bearing mice. Mechanistically, we found that ethonafide inhibits topoisomerase II activity in human prostate cancer cell lines and equally inhibits purified topoisomerase IIα and recombinant topoisomerase IIβ. The inhibition of topoisomerase II activity was due to stabilization of the cleavable complex, involving both topoisomerase IIα and β. By creating stable DU 145 cell lines with decreased expression of either topoisomerase IIα or β, we found that topoisomerase IIα is necessary for ethonafide-induced cytotoxicity. The decrease in sensitivity to ethonafide was due to a decrease in DNA damage and an increase in DNA repair as measured by the neutral comet assay. Additionally, ethonafide induces potent G₂ cell cycle arrest in the DU 145 human prostate cancer cell line. Ethonafide also induces apoptosis as measured by procaspase and PARP cleavage. In conclusion, we have identified ethonafide as a topoisomerase II poison and determined that it is topoisomerase IIα-specific in the DU 145 human prostate cancer cell line. Due to ethonafide’s activity in vitro and in vivo, decreased toxicity in mice compared to mitoxantrone, and its activity in multi-drug resistant cancer cell lines, ethonafide may be a suitable replacement to mitoxantrone for the treatment of hormone-refractory prostate cancer.