Doxorubicin (DOX) is one of the most effective chemotherapeutic agents for the treatment of a number of malignancies. However, its use is limited by serious cardiotoxic effects, for which there are currently no reliable pharmacologic therapies. Estrogen has exhibited protective effects against cardiac stressors in male and female animal models; however, its effects on DOX‑induced cardiotoxicity remain unknown. High mortality and morbidity rates have been observed in patients with cancer worldwide, and DOX is often administered to a greater number of men than women. Therefore, the present study employed male Sprague-Dawley rats to evaluate the protective effects of 17β-estradiol (E2) against DOX-induced cardiotoxicity. A total of 4 mg/kg DOX was administered to 14‑week‑old male Sprague‑Dawley rats by intraperitoneal injection twice a week for 2 weeks. At 3 weeks following the first injection of DOX, an echocardiographic study revealed that DOX administration significantly decreased cardiac ejection fraction and fractional shortening by 20 and 29%, respectively, when compared with the vehicle‑treated control rats (P<0.05). This was associated with decreased heart weight, myofibrillar disorganization and myofiber loss. The serum biomarkers for heart injury, including alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and creatine kinase, were increased in DOX vs. vehicle‑treated rats (P<0.05). E2 treatment by a daily subcutaneous injection of 2 mg/kg body weight attenuated the cardiotoxic effects of DOX. In addition, E2 treatment inhibited the DOX‑induced increase in the expression of cardiac genes, nicotinamide adenine dinucleotide phosphate oxidase (NOX) 2, NOX4, B‑cell lymphoma 2‑associated X protein and caspase 3. These results demonstrate that E2 treatment may protect the heart against DOX-induced cardiotoxicity in male rats potentially through the regulation of NOX2, NOX4 and apoptosis genes.