Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress (laminar flow). Laminar shear flow, which is atheroprotective, increases nitric oxide (NO) production via the activation of endothelial nitric oxide synthase (eNOS). Recent study suggests that NO tends to react on catalytic Cys residue of protein tyrosine phosphatase (PTP) that transiently inhibits PTP’s activity and alters signaling pathways. In the present study, we investigate the NO-mediated S-nitrosylation of PTP in ECs exposed to flow. Cultured ECs were exposed to laminar flow by a parallel-plate flow chamber system. Laminar flow to ECs leads to an elevated and sustained eNOS activation. Shear flow to ECs, similar to SNAP (NO donor) treatment, transiently inhibited PTPs activities via S-nitrosylation by eNOS-derived NO. This S-nitrosylation of PTP appears to be time- and shear force- dependent. Utilizing biotin switch method, a sustained increase of S-nitrosylation was detected in endogenous PTPs including SHP2. ECs over-expressed SHP2 mutant on catalytic site (SHP2C459S), but not other cysteine residue, showed an emphatic decrease of S-nitrosylation indicating that catalytic Cys residue in PTP is essential for S-nitrosylation in ECs under flow. Our results indicate that an elevated NO levels in ECs under flow predominantly S-nitrosylated the catalytic Cys residue in PTP. S-nitrosylation of catalytic Cys residue in PTP may protect it from H2O2-induced irreversible oxidative modification. This reversible S-nitrosylation of PTP may play a crucial role in modulating endothelial responses to various stimuli under physiological flow condition.