Connexins (Cxs) are a group of integral membrane proteins which can form gap junctions between adjacent cells. Recently, it was reported that Cx43 is expressed not only in the plasma membrane, but also in the inner mitochondrial membrane, and that it regulates mitochondrial functions. Cx40 is predominantly expressed in vascular endothelial cells (ECs) and plays an important role in the electrical propagation between ECs and endothelial/smooth muscle cells. However, it is unknown whether Cx40 is expressed in the mitochondria and what the role of mitochondrial Cx40 is in endothelial functions. We observed in coronary ECs that Cx40 protein was expressed in the mitochondria, as determined by Western blot and immunofluorescence studies. We found that mouse coronary ECs (MCECs) isolated from Cx40 knockout (Cx40 KO) mice exhibited significantly lower resting mitochondrial calcium concentration ([Ca²⁺]_mito) than MCECs from wild-type (Wt) mice. After increasing the cytosolic Ca²⁺ concentration ([Ca²⁺]_cyto) using cyclopiazonic acid, calcium uptake into the mitochondria was significantly attenuated in MCECs from Cx40 KO mice compared to Wt MCECs. There was no difference in the resting [Ca²⁺]_cyto and store-operated calcium entry in MCECs from Wt and Cx40 KO mice. We also detected a significant decrease in the concentration of mitochondrial reactive oxygen species (ROS) in Cx40 KO MCECs. Cx40 overexpression in ECs significantly increased resting [Ca²⁺]_mito level and calcium uptake by mitochondria in response to increased [Ca²⁺]_cyto and augmented mitochondrial ROS production. These data suggest that mitochondrial Cx40 contributes to the regulation of mitochondrial calcium homeostasis.