The AMP-activated protein kinase (AMPK) and glycogen are essential for maintaining metabolic homeostasis. The energy-sensing AMPK heterotrimer contains a regulatory β subunit with a carbohydrate-binding module (CBM) known to bind glycogen. However, the physiological roles of AMPK-glycogen binding in metabolism in vivo are unknown. To determine the effects of disrupting AMPK-glycogen binding, two whole-body knock-in (KI) mouse lines were generated targeting tryptophan residues known to mediate glycogen binding in either the AMPK β1 (W100A KI) or β2 (W98A KI) subunit, predominantly expressed in liver and skeletal muscle, respectively. Whole-body, serum and tissue analyses were performed in male KI and wild type (WT) litter mate mice maintained on an ad libitum chow diet. Intraperitoneal glucose tolerance testing revealed normal glucose tolerance in W100A mice but impaired glucose handling in W98A mice (56% increase in AUC; P<0.05) compared to WT, with no differences observed in fasting serum insulin. Body composition (determined from EchoMRI) showed normal whole-body fat and lean mass in W100A mice. Strikingly, W98A mice displayed a 42% increase in fat mass (P<0.05) and 5% decrease in lean mass (P<0.05) relative to WT. Metabolic caging demonstrated no changes in cumulative food intake, O2 consumption, indirect calorimetry or infrared-based activity levels. No differences were observed in resting tissue glycogen content, epididymal fat pad mass, hindlimb muscle mass or liver and muscle mitochondrial content between KI and respective WT mice. Fat deposition was increased by 61% in W100A liver (P<0.05) and 54% in W98A quadricep muscle (P<0.05) versus WT, concomitant with reductions in total protein content of AMPK α and β subunits. These data suggest that glycogen-bound AMPK is central to whole-body and tissue metabolism. We reveal new insights into the physiological roles of AMPK and suggest that loss of glycogen binding negatively impacts metabolism via reductions in the cellular AMPK pool.