Insulin resistance, a major factor in the development of type 2 diabetes, is known to be associated with defects in blood vessel relaxation. The role of Akt on insulin-induced relaxation of vascular smooth muscle cell (VSMC) was investigated using siRNA targeting Akt (siAKTc) and adenovirus constructing myristilated Akt to either suppress endogenous Akt or overexpress constitutively active Akt, respectively. siAKTc decreased both basal and insulin-induced phosphorylations of Akt and glycogen synthase kinase 3beta, abolishing insulin-induced nitric oxide synthase (iNOS) expression. cGMP-dependent kinase 1alpha (cGK1alpha) and myosin-bound phosphatase (MBP) activities, both downstream of iNOS, were also decreased. siAKTc treatment resulted in increased insulin and ANG II-stimulated phosphorylation of contractile apparatus, such as MBP substrate (MYPT1) and myosin light chain (MLC20), accompanied by increased Rho-associated kinase alpha (ROKalpha) activity, demonstrating the requirement of Akt for insulin-induced vasorelaxation. Corroborating these results, constitutively active Akt upregulated the signaling molecules involved in insulin-induced relaxation such as iNOS, cGK1alpha, and MBP activity, even in the absence of insulin stimulation. On the contrary, the contractile response involving the phosphorylation of MYPT1 and MLC20, and increased ROKalpha activity stimulated by ANG II were all abolished by overexpressing active Akt. In conclusion, we demonstrated here that insulin-induced VSMC relaxation is dependent on Akt activation via iNOS, cGK1alpha, and MBP activation, as well as the decreased phosphorylations of MYPT1 and MLC20 and decreased ROKalpha activity.