Doxorubicin (DOX) is a potent and broad-spectrum anthracycline chemotherapeutic agent, but dose-dependent cardiotoxic side effects limit its clinical application. This toxicity is closely associated with the generation of reactive oxygen species (ROS) radical during DOX metabolism. The present study investigated the effects of Berberine (Ber) on DOX-induced acute cardiac injury in a rat model and analysed its mechanism in cardiomyocytes in vitro. Serum creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and malondialdehyde (MDA) levels were significantly increased in the DOX group compared with the control group. This increase was accompanied by cardiac histopathological injury and a decrease in cardiomyocyte superoxide dismutase (SOD) and catalase (CAT). CK, CK-MB and MDA levels decreased and SOD and CAT levels increased in the Ber-treated group compared to the DOX group. Ber ameliorated the DOX-induced increase in cytosolic calcium concentration ([ Ca2+] i), attenuated mitochondrial Ca2+ overload and restored the DOX-induced loss of mitochondrial membrane potential in vitro. These results demonstrated that Ber exhibited protective effects against DOX-induced heart tissue free radical injury, potentially via the inhibition of intracellular Ca2+ elevation and attenuation of mitochondrial dysfunction.