AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by suppressing anabolic processes and activating catabolic processes. AMPK activators are an important therapeutic target for metabolic syndrome due to favorable physiological effects of AMPK activation on metabolism. Recent studies show that niclosamide, an FDA-approved anthelmintic drug that exerts an uncoupling effect on the mitochondria of the parasite, improves blood glucose levels and reduces hepatic steatosis in mice via AMPK activation. Niclosamide is thought to activate AMPK by increasing AMP/ATP ratio through mitochondrial uncoupling, but details of its action remain unclear. In this study, we found that niclosamide also activates the AMPK complex, which contains the AMP-insensitive g subunit. Further, niclosamide shows greater AMPK activation for the AMPK complex containing 132 subunit, but not the 131 subunit. This effect was inhibited by substituting the Ser108 residue of the 132 subunit to alanine. Niclosamide displays a novel AMPK activation mechanism independent of the increase in AMP/ATP ratio. (C) 2020 Elsevier Inc. All rights reserved.