The AMP-activated protein kinase (AMPK) is known to modulate the expression of mitochondrial and glucose utilising genes but the mechanisms are not well understood. In endothelial cells adenoviral overexpression of the AMPKa2 subunit resulted in the modulation of approximately 700 genes including the endothelial nitric oxide synthase (eNOS) and the transcription factor Krueppel-like factor-2 (KLF2) which is crucial for the shear stress dependent increase of eNOS expression. In murine aorta as well as in cultured cells deletion or inhibition of the expression of AMPKa2 resulted in a significantly increased eNOS protein expression. The NO-dependent vasodilation in response to bradykinin was increased in the perfused hindlimb from AMPKa2^-/- animals compared to wild type littermates. As acetylation of histones is crucial for the cell type specific eNOS expression we analyzed the effect of AMPK on the histone acetylase P300. In in vitro kinase assays P300 was phosphorylated by AMPK. Also P300 protein expression was significantly decreased in aortic lysates from AMPKa2^-/- mice. Inhibition of P300 by curcumin and downregulation of P300 with siRNA resulted in an increase in eNOS protein expression in HUVEC. To investigate if P300 attenuates eNOS expression by interference with KLF2 we used lentivirally infected HUVEC constitutively overexpressing KLF2. In these cells the effect of the P300 siRNA on the increase of eNOS expression was slightly enhanced. In summary these data suggest that AMPKa2 attenuates P300 activity and one consequence of this inhibition is the interference with KLF2 activity and a subsequent attenuation of eNOS expression.