Sphingosine 1-phosphate (S1P) rapidly increases endothelial barrier function and induces the assembly of adherens junction proteins, VE-cadherin and catenins. Since VE-cadherin contributes to the stabilization of the endothelial barrier, we determined if the rapid, barrier-enhancing activity of S1P requires VE-cadherin. Ca²⁺-dependent, homophilic VE-cadherin binding of endothelial cells, derived from human umbilical veins and grown as monolayers, was disrupted with EGTA, an antibody to the extracellular domain of VE-cadherin, or gene silencing of VE-cadherin with small interfering RNA (siRNA). All three protocols caused a reduction in the immunofluorescent localization of VE-cadherin at intercellular junctions, the separation of adjacent cells, and a decrease in basal, endothelial electrical resistance. In all three conditions, S1P rapidly increased endothelial electrical resistance. These findings demonstrate that S1P enhances the endothelial barrier independently of homophilic VE-cadherin binding. Junctional localization of VE-cadherin, however, was associated with the sustained activity of S1P. Imaging with phase-contrast and differential interference-contrast (DIC) optics revealed that S1P induced cell spreading and closure of intercellular gaps. Pretreatment with Latrunculin B, an inhibitor of actin polymerization, or Y-27632, a Rho kinase inhibitor, attenuated cell spreading and the rapid increase in electrical resistance induced by S1P. We conclude that S1P rapidly closes intercellular gaps, resulting in an increased electrical resistance across endothelial cell monolayers, via cell spreading and Rho kinase and independently of VE-cadherin.