Doxorubicin (DOX) is one of the most important anticancer agents used in treating breast cancer. However, chronic cardiotoxicity and multidrug resistance limit the chemotherapeutic use of DOX. This study aimed to evaluate the capability of calcium channel blocker diltiazem (DIL) to reverse DOX resistance in breast cancer MCF-7 cells and to confer protection against DOX-induced cardiotoxicity in Wistar rats. For this purpose, we explored the effects of DOX on cell cycle phase distribution and expression of ABCB1, FOXO3a, and p53 genes in the presence and absence of DIL (20 μg/ml) and studied the ability of DIL to prevent DOX-induced cardiotoxicity after a single injection of DOX (15 mg/kg) in male Wister rats. We found that compared with DOX alone treatment, DIL + DOX treatment down regulated the ABCB1 gene expression by > fourfold but up regulated the FOXO3a and p53 genes expression by 1.5 fold. DIL treatment conferred protection against DOX-induced cardiotoxicity, as indicated by a decrease in the levels of the cardiac enzyme creatine kinase MB and malondialdehyde and an increase in the total antioxidant capacity and glutathione peroxidase levels. These biochemical results were further confirmed by the histopathological investigation of cardiac cells, which showed normal cardiac cells with central vesicular nuclei and prevention of DOX-induced disruption of normal cardiac architecture in the DIL to DOX group. Taken together, our results indicate that DIL treatment can reverse the resistance of breast cancer cells to the therapeutic effects of DOX and can protect against DOX-induced cardiotoxicity in rats.