Carvedilol, a nonselective β-adrenergic receptor blocker, has been reported to improve endothelial dysfunction in patients with cardiovascular diseases and in a diabetic animal model but that mechanism of action is unknown. The purpose of this study was to investigate the effect of carvedilol on the endothelial-response of aortas from diabetic mice and the underlying mechanism. Vascular reactions and protein expressions were measured in aortas isolated from both control and nicotinamide and streptozotocin-induced diabetic mice (DM). UK14304 (UK)-induced endothelial-dependent relaxation declined along with the decrease of nitric oxide (NO) levels in aortas from DM. Carvedilol prevented the inhibition of UK-induced relaxation and NO production caused by DM. The phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) were very low in UK-stimulated DM aortas compared with those of control group. Treatment with carvedilol significantly increased not only the phosphorylation of Akt and eNOS under UK-stimulation but also the AMP-activated protein kinase (AMPK) phosphorylation in the DM. In conclusion, carvedilol significantly ameliorated the endothelial dysfunction in DM aortas, in which increased NO levels through Akt/eNOS activation, up-regulated AMPK phosphorylation may be involved.