In vascular endothelial cells, store-operated calcium entry (SOCE) activates endothelial NO synthase (eNOS) and regulates nitric oxide (NO) production as well as flow-dependent mechanical stimuli. Stromal interaction molecule 1, or STIM1, was recently identified to be essential for SOCE, acting as a calcium sensor for intracellular calcium stores. However, how STIM1 affects endothelial function and blood pressure (BP) remains unclear. We generated STIM1 fl/fl mice and vascular endothelial cell-specific STIM1 knockout mice using the Cre–loxP system, and conducted experiments using these mice to clarify the physiological role of STIM1 in vascular endothelial function and BP as follows: (1) SOCE was analyzed in isolated aortic endothelial cells by calcium add-back with fluorescent Ca²⁺ indicators. Phosphorylation of eNOS and NO production were evaluated by immunoblotting and the NO indicator, respectively. (2) Tension of aortic rings was measured in 10-week-old mice in response to acetylcholine. (3) BP was measured in 10-week-old mice by the telemetry system. The results were: (1) SOCE, eNOS activation, and NO production were suppressed by ~50–60% in endothelial cells from STIM1 knockout. (2) Endothelium-dependent vasodilation was decreased in aortic rings from STIM1 knockout mice, whereas endothelium-independent relaxation was not altered. (3) STIM1 knockout mice exhibited significant BP elevation, especially in nighttime. (124.3 ± 2.5/99.2 ± 3.9 vs. 114.1 ± 3.2/83.6 ± 1.7 (nighttime, mmHg), 109.7 ± 1.7/83.0 ± 3.0 vs. 104.8 ± 3.3/73.7 ± 1.6 (daytime, mmHg), knockout vs. control, respectively). In conclusion, STIM1 in vascular endothelial cell modulates vascular function through NO production and has a major role in regulating BP, especially in the active time.