Endothelial cell dysfunction is implicated in cardiovascular diseases including diabetes. The decrease in nitric oxide (NO) bioavailability is the hallmark of endothelial dysfunction and it leads to attenuated vascular relaxation and atherosclerosis followed by the decrease in blood flow. In the heart, decreased coronary blood flow is responsible for insufficient oxygen supply to cardiac myocytes, and subsequently increases the incidence of cardiac ischemia. In this study, we investigate whether and how reactive oxygen species (ROS) in mitochondria contributes to coronary endothelial dysfunction in type 2 diabetic mice. Type 2 diabetic mice were induced by a high-fat diet combined with a single injection of low dose streptozotocin. Acetylcholine (ACh)-induced vascular relaxation was significantly attenuated in diabetic coronary arteries (CAs) compared to control CAs. The pharmacological approach reveals that NO-dependent relaxation, but not hyperpolarization- or prostacyclin- dependent relaxation, was decreased in CAs of diabetic mice. Attenuated ACh-induced relaxation in diabetic CAs was restored by treatment of mitoTempol (a mitochondria-specific superoxide anion scavenger) toward control level. Mouse coronary endothelial cells (MCECs) isolated from type 2 diabetic mice exhibited a significant increase in mitochondrial ROS concentration and decreased in superoxide dismutase 2 (SOD2) protein expression compared with control MCECs. Furthermore, protein ubiquitination of SOD2 was significantly increased in MCECs isolated from diabetic mice. These results suggest that augmented SOD2 ubiquitination leads to the increase in mitochondrial ROS concentration in diabetic MCECs, and attenuates coronary vascular relaxation in Type 2 diabetic mice.