Increased pulmonary endothelial cGMP was shown to prevent endothelial barrier dysfunction through activation of protein kinase G (PKG_I). Vasodilator-stimulated phosphoprotein (VASP) has been hypothesized to mediate PKG_I barrier protection because VASP is a cytoskeletal phosphorylation target of PKG_I expressed in cell-cell junctions. Unphosphorylated VASP was proposed to increase paracellular permeability through actin polymerization and stress fiber bundling; a process inhibited by PKG_I-mediated phosphorylation of Ser¹⁵⁷ and Ser²³⁹ . To test this hypothesis, we examined the role of VASP in the transient barrier dysfunction caused by H₂O₂ in human pulmonary artery endothelial cell (HPAEC) monolayers studied without and with PKG_I expression introduced by adenoviral infection (Ad.PKG). In the absence of PKG_I expression, H₂O₂ (100-250 µM) caused a transient increased permeability and pSer¹⁵⁷ -VASP formation that were both attenuated by protein kinase C inhibition. Potentiation of VASP Ser¹⁵⁷ phosphorylation by either phosphatase 2B inhibition with cyclosporin or protein kinase A activation with forskolin prolonged, rather than inhibited, the increased permeability caused by H₂O₂. With Ad.PKG infection, inhibition of VASP expression with siRNA exacerbated H₂O₂-induced barrier dysfunction but had no effect on cGMP-mediated barrier protection. In addition, expression of a Ser-double phosphomimetic mutant VASP failed to reproduce the protective effects of activated PKG_I. Finally, expression of a Ser-double phosphorylation-resistant VASP failed to interfere with the ability of cGMP/PKG_I to attenuate H₂O₂-induced disruption of VE-cadherin homotypic binding. Our results suggest that VASP phosphorylation does not explain the protective effect of cGMP/PKG_I on H₂O₂-induced endothelial barrier dysfunction in HPAEC.