Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a heterotrimeric complex that senses intracellular energy status and exerts rapid regulation on energy-demanding and -consuming metabolic pathways. Although alterations in the intracellular adenosine nucleotide pool are traditionally assumed to be the consequence of changes in energy metabolism, in this study we have addressed the question of whether extracellular adenosine contributes to AMPK regulation. In the intestinal rat epithelial cell line IEC-6, addition of adenosine rapidly increases AMP intracellular concentrations and upregulates alpha1AMPK, thus promoting phosphorylation of its downstream target acetyl-CoA carboxylase (ACC). The effect of adenosine on AMPK signaling is completely blocked by transducing IEC-6 cells with an adenoviral vector expressing a mutated alpha1 subunit, resulting in a dominant-negative effect on endogenous AMPK activity. These effects are blocked by 5'-iodotubercidine (5'-ITU), an inhibitor of adenosine kinase. Moreover, inhibition of adenosine transport through the concentrative adenosine plasma membrane transporter CNT2 with formycin B results in the blockade of adenosine-mediated AMPK signaling. Extracellular adenosine is equally able to activate AMPK and promote ACC phosphorylation in liver parenchymal cell models in a manner that is also inhibited by 5'-ITU. In summary, this study shows that adenosine, when added at physiological concentrations, activates AMPK and promotes ACC phosphorylation. Adenosine must be transported and phosphorylated to exert its action. Thus, nucleoside transporters might be novel players in the complex regulation of AMPK and energy metabolism.