Objective— Endothelial sphingosine-1-phosphate (S1P) receptor-1 (S1P ₁ ) affects different vascular functions, including blood vessel maturation and permeability. Here, we characterized the role of the zS1P ₁ ortholog in vascular development in zebrafish. Methods and Results— zS1P ₁ is expressed in dorsal aorta and posterior cardinal vein of zebrafish embryos at 24 to 30 hours postfertilization. zS1P ₁ downregulation by antisense morpholino oligonucleotide injection causes early pericardial edema, lack of blood circulation, alterations of posterior cardinal vein structure, and late generalized edema. Also, zS1P ₁ morphants are characterized by downregulation of vascular endothelial cadherin ( VE-cadherin ) and Eph receptor EphB4a expression and by disorganization of zonula occludens 1 junctions in posterior cardinal vein endothelium, with no alterations of dorsal aorta endothelium. VE-cadherin knockdown results in similar vascular alterations, whereas VE-cadherin overexpression is sufficient to rescue venous vascular integrity defects and EphB4a downregulation in zS1P ₁ morphants. Finally, S1P ₁ small interfering RNA transfection and the S1P ₁ antagonist (R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146) cause EPHB4 receptor down-modulation in human umbilical vein endothelial cells and the assembly of zonula occludens 1 intercellular contacts is prevented by the EPHB4 antagonist TNYL-RAW peptide in these cells. Conclusion— The data demonstrate a nonredundant role of zS1P ₁ in the regulation of venous endothelial barrier in zebrafish and identify a S1P ₁ / VE-cadherin / EphB4a genetic pathway that controls venous vascular integrity.