Sphingosine 1-phosphate (Sph-1-P), a biologically active lipid released from activated platelets, was found to enhance endothelial cell barrier integrity. Sph-1-P increased transendothelial electrical resistance (TER) in both human and bovine pulmonary artery and lung microvascular endothelial cells in a dose-dependent manner. This effect was reversible and dose-dependent, and it also reversed barrier dysfunction caused by thrombin. The mechanism of Sph-1-P-mediated barrier enhancement involved Gαi-receptor coupling to Edg receptors (Edg-1 and Edg-3), Rho kinase and tyrosine kinase-dependent activation, and actin filament rearrangement. Sph-1-P-induced TER enhancement was associated with Rac GTPase- and p21-associated kinase-dependent endothelial cortical actin assembly and recruitment of cofilin. These findings suggest that Sph-1-P may act late in angiogenesis to stabilize newly formed vessels by promoting cytoskeletal rearrangement and enhancing endothelial barrier integrity.Sphingosine 1-phosphate (Sph-1-P), a biologically active lipid released from activated platelets, was found to enhance endothelial cell barrier integrity. Sph-1-P increased transendothelial electrical resistance (TER) in both human and bovine pulmonary artery and lung microvascular endothelial cells in a dose-dependent manner. This effect was reversible and dose-dependent, and it also reversed barrier dysfunction caused by thrombin. The mechanism of Sph-1-P-mediated barrier enhancement involved Gαi-receptor coupling to Edg receptors (Edg-1 and Edg-3), Rho kinase and tyrosine kinase-dependent activation, and actin filament rearrangement. Sph-1-P-induced TER enhancement was associated with Rac GTPase- and p21-associated kinase-dependent endothelial cortical actin assembly and recruitment of cofilin. These findings suggest that Sph-1-P may act late in angiogenesis to stabilize newly formed vessels by promoting cytoskeletal rearrangement and enhancing endothelial barrier integrity.