We previously reported that oxidative stress induced by long-term tacrolimus treatment impairs mitochondrial function in pancreatic beta cells. In this study, we aimed to investigate the therapeutic potential of coenzyme Q10, which is known to be a powerful antioxidant, in mitochondrial dysfunction in tacrolimus-induced diabetic rats. In a rat model of tacrolimus-induced diabetes mellitus, coenzyme Q10 treatment improved pancreatic beta cell function. The administration of coenzyme Q10 improved insulin immunoreactivity within islets, which was accompanied by reductions in oxidative stress and apoptosis. Assessment of the mitochondrial ultrastructure by electron microscopy revealed that coenzyme Q10 treatment increased the size, number, and volume of mitochondria, as well as the number of insulin granules compared with that induced by tacrolimus treatment alone. An in vitro study using a pancreatic beta cell line showed that tacrolimus treatment increased apoptosis and the production of mitochondrial reactive oxygen species, while cotreatment with coenzyme Q10 effectively attenuated these alterations. At the subcellular level, tacrolimus-induced impairment of mitochondrial respiration was significantly improved by coenzyme Q10, as evidenced by the increased mitochondrial oxygen consumption and ATP production. Our data indicate that coenzyme Q10 plays an important role in reducing tacrolimus-induced oxidative stress and protects the mitochondria in pancreatic beta cells. These findings suggest that supplementation with coenzyme Q10 has beneficial effects in tacrolimus-induced diabetes mellitus.