• AMPK β subunit knock-in (KI) mice were generated to disrupt glycogen binding in vivo . • Loss of AMPK β2 glycogen binding impairs glucose handling and exercise capacity. • Loss of AMPK β2 glycogen binding increases adiposity. • AMPK β1 and β2 KI mice show increased liver and muscle fat deposition, respectively. • Loss of glycogen binding reduces cellular AMPK protein and kinase activity.