Directed cortical actin assembly is the driving force for intercellular adhesion. Regulated by phosphorylation, vasodilator-stimulated phosphoprotein (VASP) participates in actin fiber formation. We screened for endothelial proteins, which bind to VASP, dependent on its phosphorylation status. Differential proteomics identified II-spectrin as such a VASP-interacting protein. II-Spectrin binds to the VASP triple GP5-motif via its SH3 domain. cAMP-dependent protein kinase–mediated VASP phosphorylation at Ser157 inhibits II-spectrin–VASP binding. VASP is dephosphorylated upon formation of cell–cell contacts and in confluent, but not in sparse cells, II-spectrin colocalizes with nonphosphorylated VASP at cell–cell junctions. Ectopic expression of the II-spectrin SH3 domain at cell–cell contacts translocates VASP, initiates cortical actin cytoskeleton formation, stabilizes cell–cell contacts, and decreases endothelial permeability. Conversely, the permeability of VASP-deficient endothelial cells (ECs) and microvessels of VASP-null mice increases. Reconstitution of VASP-deficient ECs rescues barrier function, whereas II-spectrin binding-deficient VASP mutants fail to restore elevated permeability. We propose that II-spectrin–VASP complexes regulate cortical actin cytoskeleton assembly with implications for vascular permeability.