Micro- and macrovascular complications are commonly seen in diabetic patients and endothelial dysfunction contributes to the development and progression of the complications. Abnormal functions in endothelial cells lead to the increase in vascular tension and atherosclerosis, followed by systemic hypertension as well as increased incidence of ischemia and stroke in diabetic patients. Mitochondria are organelles serving as a source of energy production and as regulators of cell survival (e.g., apoptosis and cell development) and ion homeostasis (e.g., H+, Ca2+). Endothelial mitochondria are mainly responsible for generation of reactive oxygen species (ROS) and maintaining the Ca2+ concentration in the cytosol. There is increasing evidence that mitochondrial morphological and functional changes are implicated in vascular endothelial dysfunction. Enhanced mitochondrial fission and/or attenuated fusion lead to mitochondrial fragmentation and disrupt the endothelial physiological function. Abnormal mitochondrial biogenesis and disturbance of mitochondrial autophagy increase the accumulation of damaged mitochondria, such as irreversibly depolarized or leaky mitochondria, and facilitate cell death. Augmented mitochondrial ROS production and Ca2+ overload in mitochondria not only cause the maladaptive effect on the endothelial function, but also are potentially detrimental to cell survival. In this article, we review the physiological and pathophysiological role of mitochondria in endothelial function with special focus on diabetes.