Sphingosine 1-phosphate (Sph-1-P), a bioactive lipid released from activated platelets, enhances endothelial cell barrier integrity through Edg receptor-dependent cytoskeletal rearrangement. This study demonstrates that Sph-1-P increases transendothelial electrical resistance (TER) in human and bovine endothelial cells, indicating improved barrier function. Sph-1-P also reverses barrier dysfunction caused by thrombin, an edemagenic agent. The protective effect of Sph-1-P is mediated through Gαi-coupled Edg receptors (Edg-1 and Edg-3), Rho kinase and tyrosine kinase activation, and actin filament rearrangement. Sph-1-P promotes Rac GTPase and p21-associated kinase-dependent endothelial cortical actin assembly, leading to the recruitment of the actin filament regulatory protein cofilin. Platelet-released Sph-1-P, linked to Rac- and Rho-dependent cytoskeletal rearrangement, may act late in angiogenesis to stabilize newly formed vessels, which often display abnormally increased vascular permeability. Sph-1-P enhances endothelial cell barrier properties by activating signaling pathways involving Edg receptors, Gαi proteins, and cytoskeletal remodeling. These findings suggest that Sph-1-P plays a critical role in maintaining vascular barrier integrity and in the regulation of angiogenesis. The study highlights the importance of Sph-1-P in vascular homeostasis and its potential as a therapeutic target in diseases involving vascular dysfunction.Sphingosine 1-phosphate (Sph-1-P), a bioactive lipid released from activated platelets, enhances endothelial cell barrier integrity through Edg receptor-dependent cytoskeletal rearrangement. This study demonstrates that Sph-1-P increases transendothelial electrical resistance (TER) in human and bovine endothelial cells, indicating improved barrier function. Sph-1-P also reverses barrier dysfunction caused by thrombin, an edemagenic agent. The protective effect of Sph-1-P is mediated through Gαi-coupled Edg receptors (Edg-1 and Edg-3), Rho kinase and tyrosine kinase activation, and actin filament rearrangement. Sph-1-P promotes Rac GTPase and p21-associated kinase-dependent endothelial cortical actin assembly, leading to the recruitment of the actin filament regulatory protein cofilin. Platelet-released Sph-1-P, linked to Rac- and Rho-dependent cytoskeletal rearrangement, may act late in angiogenesis to stabilize newly formed vessels, which often display abnormally increased vascular permeability. Sph-1-P enhances endothelial cell barrier properties by activating signaling pathways involving Edg receptors, Gαi proteins, and cytoskeletal remodeling. These findings suggest that Sph-1-P plays a critical role in maintaining vascular barrier integrity and in the regulation of angiogenesis. The study highlights the importance of Sph-1-P in vascular homeostasis and its potential as a therapeutic target in diseases involving vascular dysfunction.