The AMP-activated protein kinase (AMPK) is a central regulator of cellular energy balance and metabolism. AMPK possesses a regulatory β subunit which binds glycogen, the primary storage form of glucose in liver and skeletal muscle. AMPK-glycogen binding has been proposed as a cellular energy sensing mechanism; however, the physiological significance of this binding in vivo remains unknown. We generated novel whole-body AMPK double knock-in (DKI) mice with critical tryptophan residues in both AMPK β subunit isoforms that mediate glycogen binding substituted with alanine to determine the effects of disrupting AMPK-glycogen binding on whole-body glucose homeostasis and tissue metabolism. EchoMRI body composition analysis, intraperitoneal glucose and insulin tolerance testing (IPGTT and IPITT, respectively) and serum and tissue biochemical analyses were performed in chow-fed male AMPK DKI and wild type (WT) litter mate control mice. DKI mice had increased whole-body, fat and lean mass versus WT (n=10-22). DKI mice displayed normal fed and fasting blood glucose levels with no changes in IPGTT (n=9-11) and IPITT (n=11-12) blood glucose responses relative to WT, despite significantly elevated fasting serum insulin concentration (n=9-12). There were no differences between genotypes in GLUT4 or in total or phosphorylated Akt or AS160 protein content in skeletal muscle in the fed or fasted state (n=4-6). DKI mice displayed decreased liver AMPKα protein content in fed and fasted conditions. AMPK Thr172 phosphorylation was unaltered in the fed state but was increased in fasted DKI mice compared to WT. However, DKI mice displayed no differences in total or Ser79 phosphorylation of acetyl-CoA carboxylase, a downstream AMPK substrate, in either the fed or fasted state compared to WT. Together, these findings establish physiological roles of AMPK-glycogen binding in mice and suggest that disrupting binding in vivo increases whole-body fat and lean mass and promotes hyperinsulinemia via reducing tissue AMPK.