AMP-activated protein kinase (AMPK) is an evolutionary conserved serine/threonine protein kinase central to the regulation of energy balance at both the cellular and whole-body level. AMPK has a heterotrimeric structure, consisting of a catalytic alpha-subunit and regulatory beta- and gamma-subunits, each of which has two or more isoforms that are differentially expressed in various tissues and that arise from distinct genes. The growing realization that AMPK regulates the coordination of anabolic (synthesis and storage of glucose and fatty acids) and catabolic (oxidation of glucose and fatty acids) metabolic processes represents an attractive therapeutic target for intervention in type 2 diabetes. The stimulation of AMPK activity in muscle and liver is now seen as a likely mechanism through which anti-diabetic agents, including metformin, act to improve metabolic parameters. The importance of AMPK in vascular system is best demonstrated by recent studies showing that pharmacological AMPK activation by metformin improve endothelial function. A target of AMPK is endothelial nitric oxide synthase (eNOS), an important modulator of angiogenesis and vascular tone. By using AMPK knockout mice, it has been clearly established that AMPK associate with, and phosphorylate eNOS in endothelial cells, thus increasing eNOS activity and NO production. Thus, AMPK could trigger vasodilatation and participate in blood flow regulation. Furthermore, pharmacological activation of AMPK by AICAR induces relaxation of mouse aorta in an endothelium and eNOS-independent manner, suggesting a direct effect on smooth muscle cells. AMPK activation may be an additional mechanism by which hypoxia or metabolic challenge can induce vasorelaxation of large vessels, thereby increasing oxygen availability in peripheral tissues. AMPK appears as a new player in the complex signaling pathways that regulate vascular tone.