The serine/threonine protein kinase Akt (protein kinase B) phosphorylates endothelial cell nitric oxide synthase (eNOS) and enhances its ability to generate nitric oxide (NO). Because NO is an important regulator of vasomotor tone, we investigated whether Akt can regulate endothelium-dependent vasomotion in vivo using a rabbit femoral artery model of gene transfer. The endothelium of isolated femoral arteries was infected with replication-defective adenoviral constructs expressing β-galactosidase, constitutively-active Akt (myr-Akt), or dominant-negative Akt (dn-Akt). Femoral arteries transduced with myr-Akt showed a significant increase in resting diameter and blood flow, as assessed by angiography and Doppler flow measurements, respectively. L-NAME, an eNOS inhibitor, blocked myr-Akt–mediated vasodilatation. In contrast, endothelium-dependent vasodilatation in response to acetylcholine was attenuated in vessels transduced with dn-Akt, although these vessels showed normal responses to nitroglycerin, an endothelium-independent vasodilator. Similarly, relaxation of murine aorta ex vivo in response to acetylcholine, but not nitroglycerin, was inhibited by transduction of dn-Akt to the endothelium. These data provide evidence that Akt functions as key regulator of vasomotor tone in vivo.