Objective: Diabetic cardiomyopathy (CMP) is an important complication of long-standing hyperglycemia caused by metabolic and mitochondrial disturbance. However, the beneficial effects of resistance exercise (RE) on the prevention of diabetic CMP and cardiac mitochondrial dysfunction remain unclear. The aim of this study was to explore the effects of RE on cardiac contractility and mitochondrial dysfunction using a diabetic animal model. Methods: Fourteen Otsuka Long Evans Tokushima Fatty rats were assigned to sedentary control (SC, n = 7) and RE (n = 7) groups at 28 weeks of age. Long-Evans Tokushima Otsuka (LETO, n = 7) rats were used as non-diabetic controls. RE rats were trained by 20 repetitions of climbing a ladder 5 days per week. Results: Weight gain and metabolic alterations characteristic of SC compared with LETO were reduced in RE rats after 12 weeks of exercise. Increased left ventricular dimensions in systole and decreased functional shortening in SC rats were significantly reversed in RE rats. Collapsed sarcomeres and a decreased number of mitochondria in SC rat hearts were not observed in RE rat hearts. In addition, attenuated expression of the peroxisome proliferator-activated receptor gamma coactivator 1α and mitochondrial transcription factor A in SC were increased in RE rat hearts. Greater proton leak and increased ROS production with depolarized mitochondrial membrane potentials in SC rat hearts were attenuated with higher expression of mitochondrial superoxide dismutase 2 and lower expression of uncoupling protein 2 and 3 in RE rat hearts. Conclusion: These data suggested that RE is effective in the prevention of CMP during the course of diabetes in relation to the attenuation of metabolic disturbances and mitochondrial dysfunction, which may contribute to enhanced contractility of the diabetic heart in animal models.