At sites of ongoing inflammation, polymorphonuclear leukocytes (PMN, neutrophils) migrate across vascular endothelia, and such transmigration has the potential to disturb barrier properties and can result in intravascular fluid loss and edema. It was recently appreciated that endogenous pathways exist to dampen barrier disruption during such episodes and may provide an important anti‐inflammatory link. For example, during transmigration, PMN‐derived adenosine activates endothelial adenosine receptors and induces a cAMP‐dependent resealing of endothelial barrier function. In our study reported here, we sought to understand the link between cyclic nucleotide elevation and increased endothelial barrier function. Initial studies revealed that adenosine‐induced barrier function is tightly linked to activation of protein kinase A (PKA). Because PKA selectively phosphorylates serine and threonine residues, we screened zonula occludens‐1 (ZO‐1) immunoprecipitates for the existence of such phosphorylated proteins as targets for barrier regulation. This analysis revealed a dominantly phosphorylated band at 50 kDa. Microsequencing identified this protein as vasodilator‐stimulated phosphoprotein (VASP), an actin binding protein with multiple serine/threonine phosphorylation sites. Immunofluorescent microscopy revealed that VASP localizes to endothelial junctional complexes and colocalizes with ZO‐1, occludin, and junctional adhesion molecule‐1 (JAM‐1). To address the role of phospho‐VASP in regulation of barrier function, we generated a phosphospecific VASP antibody targeting the Ser157 residue phosphorylation site, the site preferred by PKA. Immunolocalization studies with this antibody revealed that upon PKA activation, phospho‐VASP appears at cell‐cell junctions. Transient transfection of truncated VASP fragments revealed a parallel increase in barrier function. Taken together, these studies reveal a central role for phospho‐VASP in the coordination of PKA‐regulated barrier function, such as occurs during episodes of inflammation.