Objective: Glycogen is a major energy reserve in liver and skeletal muscle. The master metabolic regulator AMP-activated protein kinase (AMPK) associates with glycogen via its regulatory β subunit carbohydrate-binding module (CBM). However, the physiological role of AMPK-glycogen binding in energy homeostasis has not been investigated in vivo. This study aimed to determine the physiological consequences of disrupting AMPK-glycogen interactions. Methods: Glycogen binding was disrupted in mice via whole-body knock-in (KI) mutation of either the AMPK β1 (W100A) or β2 (W98A) isoform CBM. Systematic whole-body, tissue and molecular phenotyping was performed in KI and respective wild-type (WT) mice. Results: While β1 W100A KI did not affect whole-body metabolism or exercise capacity, β2 W98A KI mice displayed increased adiposity and impairments in whole-body glucose handling and maximal exercise capacity relative to WT. These KI mutations resulted in reduced total AMPK protein and kinase activity in liver and skeletal muscle of β1 W100A and β2 W98A, respectively, versus WT mice. β1 W100A mice also displayed loss of fasting-induced liver AMPK total and α-specific kinase activation relative to WT. Destabilisation of AMPK was associated with increased fat deposition in β1 W100A liver and β2 W98A skeletal muscle versus WT. Conclusions: These results demonstrate that glycogen binding plays critical roles in stabilising AMPK and maintaining cellular, tissue and whole-body energy homeostasis.