Endothelial cell-cell junctions control efflux of small molecules and leukocyte transendothelial migration (TEM) between blood and tissues. Inhibitors of phosphoinositide 3-kinases (PI3Ks) increase endothelial barrier function, but the roles of different PI3K isoforms have not been addressed. In this study, we determine the contribution of each of the four class I PI3K isoforms (p110 alpha, -beta, -gamma, and -delta) to endothelial permeability and eukocyte TEM. We find that depletion of p110 alpha but not other p110 isoforms decreases TNF-induced endothelial permeability, Tyr phosphorylation of the adherens junction protein vascular endothelial cadherin (VE-cadherin), and leukocyte TEM. p110 alpha selectively mediates activation of the Tyr kinase Pyk2 and GTPase Rac1 to regulate barrier function. Additionally, p110 alpha mediates the association of VE-cadherin with Pyk2, the Rac guanine nucleotide exchange factor Tiam-1 and the p85 regulatory subunit of PI3K. We propose that p110 alpha regulates endothelial barrier function by inducing the formation of a VE-cadherin-associated protein complex that coordinates changes to adherens junctions with the actin cytoskeleton.