Doxorubicin is among the most effective and widely used anticancer drugs in the clinic. However, cardiotoxicity is one of the life-threatening side effects of doxorubicin-based therapy. Dexrazoxane (Zinecard, also known as ICRF-187) has been used in the clinic as a cardioprotectant against doxorubicin cardiotoxicity. The molecular basis for doxorubicin cardiotoxicity and the cardioprotective effect of dexrazoxane, however, is not fully understood. In the present study, we showed that dexrazoxane specifically abolished the DNA damage signal γ-H2AX induced by doxorubicin, but not camptothecin or hydrogen peroxide, in H9C2 cardiomyocytes. Doxorubicin-induced DNA damage was also specifically abolished by the proteasome inhibitors bortezomib and MG132 and much reduced in top2β^−/− mouse embryonic fibroblasts (MEF) compared with TOP2β^+/+ MEFs, suggesting the involvement of proteasome and DNA topoisomerase IIβ (Top2β). Furthermore, in addition to antagonizing Top2 cleavage complex formation, dexrazoxane also induced rapid degradation of Top2β, which paralleled the reduction of doxorubicin-induced DNA damage. Together, our results suggest that dexrazoxane antagonizes doxorubicin-induced DNA damage through its interference with Top2β, which could implicate Top2β in doxorubicin cardiotoxicity. The specific involvement of proteasome and Top2β in doxorubicin-induced DNA damage is consistent with a model in which proteasomal processing of doxorubicin-induced Top2β-DNA covalent complexes exposes the Top2β-concealed DNA double-strand breaks. [Cancer Res 2007;67(18):8839–46]