High ACE levels are a risk factor for cardiovascular disease, but little is known about the molecular mechanisms regulating ACE expression in endothelial cells. Here, we determined the role of the AMPK and the miR-143/145-cluster, since they regulate ACE expression in other cell types and are activated/upregulated in response to shear stress. Shear stress time-dependently decreased ACE expression in human endothelial cells, an effect sensitive to silencing (siRNA) of the catalytic AMPK α2 (but not the α1) subunit. The AMPK activators, AICAR and metformin also decreased ACE expression in cultured endothelial cells and endothelial cells from AMPKα2-/- (but not AMPKα1-/-) mice expressed significantly higher ACE levels. Consequently, AMPKα2-/- mice were more sensitive to the hypotensive effect of the ACE-inhibitor ramipril and demonstrated an impaired bradykinin-induced hindlimb vasodilatation. The latter response was restored by ramiprilat and was also evident in endothelial cell specific AMPKα2 knockout mice. In miR-143/145-deficient LacZ reporter gene mice, ACE was identified as target of the miR-143/145 cluster in vascular smooth muscle cells. We confirmed the suppressive effect of miR-143/145 on ACE-expression in isolated endothelial cells from these mice as well as in human endothelial cells by overexpressing miR-143/145. AMPKα2 deletion (in vitro and in vivo) decreased miR-143/145 levels and overexpression of pre-miR-143/145 decreased endothelial ACE expression. Because shear stress increased levels of premature and mature miR-143/145, without affecting the primary transcript or miR-143/145 promoter activity, it seems that AMPKα2 regulates miR expression via a post-transcriptional rather than transcriptional mechanism. Since p53 is an AMPK target, regulates miRs at the transcriptional and post-transcriptional levels and shear-stress elicited the AMPKα2-mediated phophorylation of p53 (Ser15), we focused on validating AMPK-p53 effect on miR expression. Suppression of p53 (siRNA) decreased miR-143/145 levels, increased endothelial ACE expression and prevented its shear stress-induced downregulation. Streptozotocin-induced diabetes increased the phosphorylation of the AMPK substrates, p53 and acetyl-coenzyme A carboxylase, at the same time as increasing miR-143/145 and decreasing ACE levels. In conclusion, the activation of AMPKα2 suppresses endothelial ACE expression via p53 activation and post-transcriptional upregulation of miR-143/145. Since dysregulation of AMPK as well as p53 plays a major role in the development of several diseases (e.g. diabetes, cancer), their effect on the regulation of miR-143/145 and thus ACE levels might explain the development of disease-associated cardiovascular disorders.