Protease activated receptors (PARs) are G protein-coupled receptors that are known to regulate endothelial nitric oxide synthase (eNOS) activity in part by phosphorylating the enzyme at various sites. Ser1177 is a positive regulatory site, which leads to the enhanced production of nitric oxide (NO), a vasodilator of arteries. Thr495 is a negative regulatory site, which inhibits NO production. We have shown that thrombin, a PAR agonist, mediates eNOS-Ser1177 phosphorylation through Gq and a calcium and protein kinase C (PKC)-delta sensitive, but phosphatidylinositol 3-kinase (PI3K)/Akt-independent pathway. However, the mechanism for eNOS-Thr495 phosphorylation by PAR agonists is unknown. We used a specific synthetic PAR-1 activating peptide, TFLLR, and thrombin to assess the role of PAR-1 involvement in the phosphorylation of eNOS-Thr495 in human umbilical vein endothelial cells (HUVECs). Using Western blot analysis and the Griess Reagent assay, we found that both agonists phosphorylated Thr495 in a time- and dose-dependent manner and significantly decreased nitrite production, respectively. Pretreatment of cells with the PAR-1 inhibitor, SCH-79797, resulted in a significant decrease in thrombin- and TFLLR-induced phosphorylation of eNOS-Thr495 and an increase in nitrite production. We further demonstrated that inhibition of Rho with C3 exoenzyme or dominant negative (dn) RhoA, and inhibition of Rho-Kinase (ROCK) with Y-27632 caused a significant decrease in thrombin and TFLLR-induced Thr495 phosphorylation. Blockade of the Rho/ROCK pathway also caused an increase in nitrite production. This suggests that PAR-1 regulates eNOS activity via phosphorylation of eNOS-Thr495, which is dependent upon activation of the Rho/ROCK pathway. These findings will be beneficial in further understanding the signaling pathways that regulate eNOS-induced NO production, which plays an important role in endothelial dysfunction associated with cardiovascular disease.