Rationale: Endothelial dysfunction is an early event in cardiovascular disease and characterized by reduced production of nitric oxide (NO). The F-BAR protein NO synthase traffic inducer (NOSTRIN) is an interaction partner of endothelial NO synthase and modulates its subcellular localization, but the role of NOSTRIN in pathophysiology in vivo is unclear. Objective: We analyzed the consequences of deleting the NOSTRIN gene in endothelial cells on NO production and cardiovascular function in vivo using NOSTRIN knockout mice. Methods and Results: The levels of NO and cGMP were significantly reduced in mice with endothelial cell–specific deletion of the NOSTRIN gene resulting in diastolic heart dysfunction. In addition, systemic blood pressure was increased, and myograph measurements indicated an impaired acetylcholine-induced relaxation of isolated aortic rings and resistance arteries. We found that the muscarinic acetylcholine receptor subtype M3 (M3R) interacted directly with NOSTRIN, and the latter was necessary for correct localization of the M3R at the plasma membrane in murine aorta. In the absence of NOSTRIN, the acetylcholine-induced increase in intracellular Ca ²⁺ in primary endothelial cells was abolished. Moreover, the activating phosphorylation and Golgi translocation of endothelial NO synthase in response to the M3R agonist carbachol were diminished. Conclusions: NOSTRIN is crucial for the localization and function of the M3R and NO production. The loss of NOSTRIN in mice leads to endothelial dysfunction, increased blood pressure, and diastolic heart failure.