As a gatekeeper of leukocyte trafficking the vasculature fulfills an essential immune function. We have recently shown that paracellular transendothelial lymphocyte migration is controlled by intercellular adhesion molecule 1 (ICAM-1)-mediated vascular endothelial cadherin (VEC) phosphorylation [Turowski et al., J. Cell Sci. 121, 29-37 (2008)]. Here we show that endothelial nitric oxide synthase (eNOS) is a critical regulator of this pathway. ICAM-1 stimulated eNOS by a mechanism that was clearly distinct from that utilized by insulin. In particular, phosphorylation of eNOS on S1177 in response to ICAM-1 activation was regulated by src family protein kinase, rho GTPase, Ca(2+), CaMKK, and AMPK, but not Akt/PI3K. Functional neutralization of any component of this pathway or its downstream effector guanylyl cyclase significantly reduced lymphocyte diapedesis across the endothelial monolayer. In turn, activation of NO signaling promoted lymphocyte transmigration. The eNOS signaling pathway was required for T-cell transmigration across primary rat and human microvascular endothelial cells and also when shear flow was applied, suggesting that this pathway is ubiquitously used. These data reveal a novel and essential role of eNOS in basic immune function and provide a key link in the molecular network governing endothelial cell compliance to diapedesis.