Basal forebrain neurons increase cortical blood flow by releasing acetylcholine (Ach), which stimulates endothelial cells (ECs) to produce the vasodilating gasotransmitter, nitric oxide (NO). Surprisingly, the mechanism whereby Ach induces NO synthesis in brain microvascular ECs is unknown. An increase in intracellular Ca2+concentration recruits a multitude of endothelial Ca2+-dependent pathways, such as Ca2+/calmodulin endothelial NO synthase (eNOS). The present investigation sought to investigate the role of intracellular Ca2+signaling in Ach-induced NO production in bEND5 cells, an established model of mouse brain microvascular ECs, by conventional imaging of cells loaded with the Ca2+-sensitive dye, Fura-2/AM, and the NO-sensitive fluorophore, DAF-DM diacetate. Ach induced dose-dependent Ca2+oscillations in bEND5 cells, 300 μM being the most effective dose to generate a prolonged Ca2+burst. Pharmacological manipulation revealed that Ach-evoked Ca2+oscillations required metabotropic muscarinic receptor (mAchR) activation and were patterned by a complex interplay between repetitive ER Ca2+release via inositol-1,4,5-trisphosphate receptors (InsP3Rs) and store-operated Ca2+entry (SOCE). A comprehensive real time-polymerase chain reaction analysis demonstrated the expression of the transcripts encoding for M3-mAChRs, InsP3R1 and InsP3R3, Stim1-2 and Orai2. Next, we found that Ach-induced NO production was hindered by L-NAME, a selective NOS inhibitor, and BAPTA, a membrane permeable intracellular Ca2+buffer. Moreover, Ach-elicited NO synthesis was blocked by the pharmacological abrogation of the accompanying Ca2+spikes. Overall, these data shed novel light on the molecular mechanisms whereby neuronally-released Ach controls neurovascular coupling in blood microvessels.