BACKGROUND: Diabetic cardiomyopathy (DCM) is a significant cardiovascular complication in diabetic patients, and treatment regimens are limited. Rhein, a compound extracted from the herb rhubarb, was investigated in this study for its efficacy on DCM and the potential mechanism. METHODS: Streptozotocin-induced DCM mice, high-glucose (HG)-treated neonatal rat cardiomyocytes (NRCMs), and H9c2 cells with ClpP knockdown were used for the study. We performed phenotypic and molecular mechanistic studies using immunoblotting, quantitative polymerase chain reaction, transmission electron microscopy, cardiac echocardiography, and histopathological analysis. RESULTS: Rhein improved the cardiac function and myocardial fibrosis, and decreased the cross-sectional area of cardiomyocytes in the DCM mice. It also improved mitochondrial dynamic disorder as evidenced by a decreased ratio of mitochondrial fission-related proteins p-Drp1 CONCLUSIONS: Our results suggest that Rhein protects DCM by ameliorating mitochondrial dynamics disorder, inhibiting cardiomyocyte apoptosis, and myocardial hypertrophy. These protective effects of Rhein may be mediated by preventing ClpP upregulation.