We found that a natural product, Sanguinarine, directly interacts with AMPK and enhances its enzymatic activity. Cell-based assays confirmed that cellular AMPK and the downstream acetyl-CoA carboxylase (ACC) were phosphorylated after Sanguinarine treatment. Sanguinarine was shown to exclusively activate AMPK holoenzymes containing alpha 1 gamma 1 complexes, and it activated both beta 1- and beta 2-containing isotypes of AMPK. Furthermore, a docking study suggested that Sanguinarine binds AMPK at the cleft between the beta and gamma domains whereas the physiological activator, AMP, binds at the well-characterized gamma domain pocket. In summary, we report that Sanguinarine is a novel, direct activator of AMPK that binds by a unique allosteric mechanism different from that of the natural AMPK ligand, AMP, and other known AMPK activators. These studies have direct applications to the pharmacological study of AMPK activation and the potential development of new therapeutics. (C) 2011 Elsevier Inc. All rights reserved.