Endothelial membrane-bound thrombomodulin is a high affinity receptor for thrombin to inhibit coagulation. We previously demonstrated that the thrombin-thrombomodulin-complex restrains cell proliferation mediated through protease activated receptor (PAR-1). We now tested the hypothesis that thrombomodulin transduces a signal to activate the endothelial NO synthase (NOS3) and to modulate G protein-coupled receptor signaling. Cultured human umbilical vein endothelial cells were stimulated with thrombin or a mutant of thrombin that binds to thrombomodulin and has no catalytic activity on PAR-1. Thrombin and its mutant dose-dependently activated NO release at cell surface. Pre-treatment with anti-thrombomodulin antibody suppressed NO response to the mutant and to low thrombin concentration and reduced by half that to high concentration. Thrombin receptor activating peptide that only activates PAR-1 and high thrombin concentration induced marked biphasic Ca²⁺ signals with rapid phosphorylation of PLC3 and NOS3 at both serine¹¹⁷⁷ and threonine⁴⁹⁵. The mutant thrombin evoked a Ca²⁺ spark and progressive phosphorylation of Src-family kinases at tyrosine⁴¹⁶ and NOS3 at only threonine⁴⁹⁵. It activated rapid phosphatidylinositol-3 kinase-dependent NO synthesis and phosphorylation of epidermal growth factor receptor (EGFR) and calmodulin kinase II. Complete EGFR inhibition only partly reduced the activation of phospholipase C1 and NOS3. Pre-stimulation of thrombomodulin did not affect NO release but reduced Ca²⁺ responses to thrombin and histamine, suggesting cross-talks between thrombomodulin and G protein-coupled receptors. This is the first demonstration of an outside-in signal mediated by the cell-surface thrombomodulin receptor to activate NOS3 through tyrosine kinase-dependent pathway. This signaling may contribute to thrombomodulin function in thrombosis, inflammation and atherosclerosis.