Regulation of actin dynamics is critical for endothelial barrier functions. We provide evidence that the actin-binding protein vasodilator-stimulated phosphoprotein (VASP) is required for endothelial barrier maintenance. Baseline permeability was significantly increased in VASP-deficient (VASP -/-) microvascular myocardial endothelial cells (MyEnd) in the absence of discernible alterations of immunostaining for adherens and tight junctions. We tested whether VASP is involved in the endothelium-stabilizing effects of cAMP or Rac 1. Forskolin and rolipram (F/R) to increase cAMP and cytotoxic necrotizing factor 1 (CNF-1) to activate Rac 1 were equally efficient to stabilize barrier functions in VASP (-/-) and in wild-type (wt) cells. In wt, VASP was phosphorylated in response to F/R but did not localize to intercellular junctions. In contrast, CNF-1 and expression of constitutively active Rac 1 induced translocation of VASP to cell borders in wt cells where it colocalized with active Rac 1. In VASP (-/-) cells, Rac 1 activity was reduced to 0.4-fold of wt levels in controls and increased about 20-fold in response to CNF-1 compared to 7-fold activation in wt cells. Moreover, inactivation of Rac 1 by lethal toxin led to a greater increase of permeability compared to wt cells. All these data suggest that VASP is involved in the regulation of Rac 1 activity. Taken together, our study indicates that VASP at least in part stabilizes endothelial barrier functions by control of Rho-family GTPases.