It has been argued whether insulin accelerates or prevents atherosclerosis. Although results from in vitro studies have been conflicting, recent in vivo mice studies demonstrated anti-atherogenic effects of insulin. Insulin is a known activator of endothelial nitric oxide synthase (NOS), leading to increased production of NO, which has potent anti-atherogenic effects. We aimed to examine the role of NOS in the protective effects of insulin against atherosclerosis. Male apolipoprotein E-null mice (8wk old) fed high cholesterol diet (1.25% cholesterol) were assigned to the following 12wk treatments: control, insulin (0.05U/day via subcutaneous pellet), N-Nitro-L-arginine methyl ester hydrochloride (L-NAME, via drinking water at 100mg/l), and insulin plus L-NAME. Insulin reduced atherosclerotic plaque burden in the descending aorta by 42% compared to control (plaque area / aorta lumen area: control, 16.5±1.9%; insulin, 9.6±1.3%, p<0.05). Although insulin did not decrease plaque burden in the aortic sinus, macrophage accumulation in the plaque was decreased by insulin. Furthermore insulin increased smooth muscle actin and collagen content, and decreased plaque necrosis, consistent with increased plaque stability. In addition, insulin treatment increased plasma NO levels, decreased iNOS staining and tended to increase phosphorylated vasodilator-stimulated phosphoprotein staining in the plaques of the aortic sinus. All these effects of insulin were abolished by co-administration of L-NAME, while L-NAME alone showed no effect. Insulin also tended to increase phoshorylated endothelial NOS and total neuronal NOS staining, effects not modified by L-NAME. In conclusion, we demonstrated that insulin treatment decreases atherosclerotic plaque burden and increases plaque stability through NOS dependent mechanisms.