Endothelial cell (EC) barrier dysfunction results in increased vascular permeability observed in inflammation, tumor angiogenesis, and atherosclerosis. The platelet‐derived phospholipid sphingosine‐1‐phosphate (S1P) decreases EC permeability in vitro and in vivo and thus has obvious therapeutic potential. We examined S1P‐mediated human pulmonary artery EC signaling and barrier regulation in caveolin‐enriched microdomains (CEM). Immunoblotting from S1P‐treated EC revealed S1P‐mediated rapid recruitment (1 µM, 5 min) to CEMs of the S1P receptors S1P₁ and S1P₃, p110 PI3 kinase α and β catalytic subunits, the Rac1 GEF, Tiam1, and α‐actinin isoforms 1 and 4. Immunoprecipitated p110 PI3 kinase catalytic subunits from S1P‐treated EC exhibited PIP₃ production in CEMs. Immunoprecipitation of S1P receptors from CEM fractions revealed complexes containing Tiam1 and S1P₁. PI3 kinase inhibition (LY294002) attenuated S1P‐induced Tiam1 association with S1P₁, Tiam1/Rac1 activation, α‐actinin‐1/4 recruitment, and EC barrier enhancement. Silencing of either S1P₁ or Tiam1 expression resulted in the loss of S1P‐mediated Rac1 activation and α‐actinin‐1/4 recruitment to CEM. Finally, silencing S1P₁, Tiam1, or both α‐actinin isoforms 1/4 inhibits S1P‐induced cortical F‐actin rearrangement and S1P‐mediated barrier enhancement. Taken together, these results suggest that S1P‐induced recruitment of S1P₁ to CEM fractions promotes PI3 kinasemediated Tiam1/Rac1 activation required for α‐actinin‐1/4‐regulated cortical actin rearrangement and EC barrier enhancement. Singleton, P. A., Dudek, S. M., Chiang, E. T., Garcia, J. G. N. Regulation of sphingosine 1‐phosphate‐induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/Rac1 and α‐actinin. FASEB J. 19, 1646–1656 (2005)