The metabolic role of 5′AMP-activated protein kinase (AMPK) in regulation of skeletal muscle metabolism in humans is unresolved. We measured isoform-specific AMPK activity and β-acetyl-CoA carboxylase (ACCβ) Ser221phosphorylation and substrate balance in skeletal muscle of eight athletes at rest, during cycling exercise for 1 h at 70% peak oxygen consumption, and 1 h into recovery. The experiment was performed twice, once in a glycogen-loaded (glycogen concentration ∼900 mmol/kg dry wt) and once in a glycogen-depleted (glycogen concentration ∼160 mmol/kg dry wt) state. At rest, plasma long-chain fatty acids (FA) were twofold higher in the glycogen-depleted than in the loaded state, and muscle α1 AMPK (160%) and α2 AMPK (145%) activities and ACCβ Ser221phosphorylation (137%) were also significantly higher in the glycogen-depleted state. During exercise, α2 AMPK activity, ACCβ Ser221phosphorylation, plasma catecholamines, and leg glucose and net FA uptake were significantly higher in the glycogen-depleted than in the glycogen-loaded state without apparent differences in muscle high-energy phosphates. Thus exercise in the glycogen-depleted state elicits an enhanced uptake of circulating fuels that might be associated with elevated muscle AMPK activation. It is concluded that muscle AMPK activity and ACCβ Ser221phosphorylation at rest and during exercise are sensitive to the fuel status of the muscle. During exercise, this dependence may in part be mediated by humoral factors.