Ca²⁺-sensing receptor (CaSR) belongs to family C of G protein-coupled receptors and is activated by the endogenous agonists such as Ca²⁺. Stimulation of CaSR expressed in vascular endothelial cells through the increase in extracellular Ca²⁺ concentration ([Ca²⁺]_o) is reported to induce vasorelaxation via the production of nitric oxide (NO). The purpose of the present study is to characterize the CaSR-mediated NO production in human vascular endothelial cells. In human endothelial EA.hy926 cells, the increase in [Ca²⁺]_o from 0.2 to 2 mM induced a concentration-dependent increase in intracellular Ca²⁺ concentration, which was significantly inhibited by NPS 2143 (a CaSR antagonist) and YM-254890 (a G_q/11 protein inhibitor). Stimulation with 2 mM Ca²⁺ for 4 h elicited an increase in the phosphorylation level of eNOS at Ser¹¹⁷⁷, which was significantly depressed by NPS 2143, YM-254890, and removal of Ca²⁺ from the medium. Ca²⁺ (2 mM) induced an increase in NO production, which was inhibited by NPS 2143, YM-254890, removal of Ca²⁺ from the medium, and L-NAME (a competitive eNOS inhibitor). These results provide evidence that activation of CaSR with extracellular Ca²⁺ facilitates NO release from human vascular endothelial cells via a G_q/11 protein-eNOS-dependent pathway.