The Src family kinases (SFKs) c-Src and Yes mediate vascular leakage in response to various stimuli including lipopolysaccharide (LPS) and VEGF. Here, we define an opposing function of another SFK, Lyn, which in contrast to other SFKs strengthens endothelial junctions and thereby restrains the increase in vascular permeability. Mice lacking Lyn displayed increased mortality in LPS-induced endotoxemia and increased vascular permeability in response to LPS or VEGF challenge as compared to wild-type littermates. Lyn knockout mice repopulated with wild-type bone marrow-derived cells have higher vascular permeability than wild-type mice, suggesting a role of endothelial Lyn in the maintenance of the vascular barrier. siRNA-mediated downregulation of Lyn disrupted endothelial barrier integrity, whereas expression of a constitutively active mutant of Lyn enhanced the barrier. However, downregulation of Lyn did not affect LPS-induced endothelial permeability. We demonstrate that Lyn association with focal adhesion kinase (FAK) and phosphorylation of FAK at tyrosine residues 576/577 and 925, were required for Lyn-dependent stabilization of endothelial adherens junctions. Thus, in contrast to c-Src and Yes, which increase vascular permeability in response to stimuli, Lyn stabilizes endothelial junctions through phosphorylation of FAK. Therefore, therapeutics activating Lyn kinase may strengthen the endothelial barrier junction and hence have anti-inflammatory potential.