VEGF is an endothelial cell cytokine that promotes angiogenesis and enhances microvascular permeability. Recently, it has been shown that the protein kinase Akt functions in a key intercellular signaling pathway downstream of VEGF. Here, we employed adenovirus‐mediated gene transfer in conjunction with the Miles assay in hairless albino guinea pigs to assess the role of Akt signaling in vascular permeability. VEGF‐induced vascular permeability was blocked by the transduction of a dominant negative mutant of Akt. Conversely, transduction of a constitutively active form of Akt promoted vascular permeability in a manner similar to VEGF protein administration. This Akt‐mediated increase in vascular permeability was inhibited by the eNOS inhibitor L‐NAME. These data show that Akt signaling is both necessary and sufficient for vascular permeability in an in vivo model.