Aconitum plants, which have analgesic, diuretic and anti-inflammatory effects, have been widely used to treat various types of disease. However, the apparent toxicity of Aconitum-derived agents, particularly in the cardiovascular system, has largely limited their clinical use. Thus, the present study investigated whether berberine (Ber), an isoquinoline alkaloid, may reduce myocardial injury induced by aconitine (AC) in rats and the underlying mechanisms. Rats (n=40) were randomly divided into four groups: Control, Chuan-wu and Chuan-wu + Ber (8 and 16 mg/kg doses). Electrocardiograms (ECG) of the rats were recorded and serum biomarkers of cardiac function [lactate dehydrogenase (LDH), creatine kinase (CK) and CK-MB] were assayed. Histopathological changes were assessed using myocardial tissue sectioning and hematoxylin and eosin staining. Additionally, the effects of Ber on AC-induced arrhythmias in rats were observed. The changes in ECG following AC perfusion were observed, and the types and onset time of arrhythmias were analyzed. Furthermore, the effects of Ber and AC on papillary muscle action potentials were observed. The results suggested that Ber ameliorated myocardial injury induced by Chuan-wu, which was indicated by reduced arrhythmias and decreased LDH, CK and CK-MB levels in serum. Furthermore, histological damage, including dilation of small veins and congestion, was also markedly attenuated by Ber. In addition, the occurrence of arrhythmias was significantly delayed, and the dosage of AC required to induce arrhythmias was also increased by Ber pretreatment. Additionally, AC-induced changes in action potential amplitude, duration of 30% repolarization and duration of 90% repolarization in the papillary muscle were attenuated by Ber. All of these results indicate that Ber had a preventive effect on acute myocardial injury induced by Chuan-wu and arrhythmias caused by AC, which may be associated with the inhibition of delayed depolarization and triggered activity caused by AC. Thus, combination treatment of Ber with Aconitum plants may be a novel strategy to prevent AC-induced myocardial injury in clinical practice.