Upon agonist stimulation, endothelial cells trigger smooth muscle relaxation through the release of relaxing factors such as nitric oxide (NO). Endothelial cells of mouse aorta are interconnected by gap junctions made of connexin40 (Cx40) and connexin37 (Cx37), allowing the exchange of signaling molecules to coordinate their activity. Wild-type (Cx40^+/+) and hypertensive Cx40-deficient mice (Cx40^−/−), which also exhibit a marked decrease of Cx37 in the endothelium, were used to investigate the link between the expression of endothelial connexins (Cx40 and Cx37) and endothelial nitric oxide synthase (eNOS) expression and function in the mouse aorta. With the use of isometric tension measurements in aortic rings precontracted with U-46619, a stable thromboxane A₂ mimetic, we first demonstrate that ACh- and ATP-induced endothelium-dependent relaxations solely depend on NO release in both Cx40^+/+ and Cx40^−/− mice, but are markedly weaker in Cx40^−/− mice. Consistently, both basal and ACh- or ATP-induced NO production were decreased in the aorta of Cx40^−/− mice. Altered relaxations and NO release from aorta of Cx40^−/− mice were associated with lower expression levels of eNOS in the aortic endothelium of Cx40^−/− mice. Using immunoprecipitation and in situ ligation assay, we further demonstrate that eNOS, Cx40, and Cx37 tightly interact with each other at intercellular junctions in the aortic endothelium of Cx40^+/+ mice, suggesting that the absence of Cx40 in association with altered Cx37 levels in endothelial cells from Cx40^−/− mice participate to the decreased levels of eNOS. Altogether, our data suggest that the endothelial connexins may participate in the control of eNOS expression levels and function.