The anthracycline doxorubicin is a widely used anticancer agent in the clinic, but its full potential cannot be realised because of dose-dependent cardiotoxicity. The mechanisms involved in this cardiotoxicity are multifactorial but the one of the major mechanisms is mediated by topoisomerase-II1. When doxorubicin is activated by formaldehyde, this results in the formation of doxorubicin-DNA adducts and enhanced levels of tumour cell kill through a non-topoisomerase-II mediated mechanism, but a corresponding lower level of cell kill to cardiomyocytes2. A variety of formaldehyde-releasing prodrugs (FRPs) were used in combination with doxorubicin to understand the cardioprotective effect in mice bearing 4T1.2 mammary tumours. In order to model doxorubicin-induced cardiac damage, an acute treatment model employing doses of doxorubicin from 4 to 16mg/kg for 72 h was chosen. Combination treatment with various FRPs was conducted in order to assess if the treatments were cardioprotective. After 72 h, a breakdown of myofibrillar structure in the doxorubicin group was observed but combination treatment employing doxorubicin and FRPs preserved the myofibrillar structure. The molecular events leading to this cardioprotection were then investigated further. Levels of covalent doxorubicin-DNA adducts in heart tissue were initially assessed. Tumour-bearing mice were injected with 14C-doxorubicin as a single agent or in combination with AN-9 (an FRP that has undergone Phase II clinical trial) or Cefditoren Pivoxil (an FRP used in the clinic). Although the 14C-doxorubicin distribution in heart tissue remains the same across the single and combination treatments, an increase of approximately 3-fold and 4-fold of adducts were detected in doxorubicin/AN-9 and doxorubicin/Cefditoren Pivoxil groups, respectively as compared to single agent doxorubicin. Transcriptomic and KEGG pathway analysis revealed that doxorubicin leads to apparent downregulation of oxidative phosphorylation and calcium signalling pathways while doxorubicin/AN-9 did not alter these pathways. In summary, combination treatment appears to alter the mechanism of action of doxorubicin which results in a cardioprotective effect.