Increased tissue permeability is commonly associated with hypoxia of many origins. Since hypoxia-inducible factor (HIF) represents a predominant hypoxia signaling mechanism, we compared hypoxia-elicited changes in tissue barrier function in mice conditionally lacking intestinal epithelial hypoxia-inducible factor-1alpha (hif1a). Somewhat surprisingly, these studies revealed that mutant hif1a mice were protected from hypoxia-induced increases in intestinal permeability in vivo. Guided by microarray analysis of tissues derived from these mutant hif1a mice, we identified HIF-1-dependent repression of vasodilator-stimulated phosphoprotein (VASP), a molecule known to be important in the control of cytoskeletal dynamics, including barrier function. Studies at the mRNA and protein level confirmed hypoxia-elicited repression of VASP in murine tissue, cultured epithelia and endothelia, as well as human saphenous vein ex vivo. Targeted repression of VASP by siRNA recapitulated our findings with hypoxia and directed overexpression of VASP abolished hypoxia-induced barrier dysfunction. Studies in the cloned human VASP promoter revealed hypoxia-dependent transcriptional repression, and functional studies by chromatin immunoprecipitation (ChIP) and site-directed mutagenesis revealed hypoxia-dependent binding of HIF-1alpha to the human VASP promoter. These studies identify HIF-1-dependent repression of VASP as a control point for hypoxia-regulated barrier dysfunction.