5′-AMP-activated protein kinase (AMPK) was recently suggested to regulate pyruvate dehydrogenase (PDH) activity and thus pyruvate entry into the mitochondrion. We aimed to provide evidence for a direct link between AMPK and PDH in resting and metabolically challenged (exercised) skeletal muscle. Compared with rest, treadmill running increased AMPKα1 activity in α₂KO mice (90%, P < 0.01) and increased AMPKα2 activity in wild-type (WT) mice (110%, P < 0.05), leading to increased AMPKα Thr¹⁷² (WT: 40%, α₂KO: 100%, P < 0.01) and ACCβ Ser²²⁷ phosphorylation (WT: 70%, α₂KO: 210%, P < 0.01). Compared with rest, exercise significantly induced PDH-E₁α site 1 (WT: 20%, α₂KO: 62%, P < 0.01) and site 2 (only α₂KO: 83%, P < 0.01) dephosphorylation and PDH_a [∼200% in both genotypes (P < 0.01)]. Compared with WT, PDH dephosphorylation and activation was markedly enhanced in the α₂KO mice both at rest and during exercise. The increased PDH_a activity during exercise was associated with elevated glycolytic flux, and muscles from the α₂KO mice displayed marked lactate accumulation and deranged energy homeostasis. Whereas mitochondrial DNA content was normal, the expression of several mitochondrial proteins was significantly decreased in muscle of α₂KO mice. In isolated resting EDL muscles, activation of AMPK signaling by AICAR did not change PDH-E₁α phosphorylation in either genotype. PDH is activated in mouse skeletal muscle in response to exercise and is independent of AMPKα2 expression. During exercise, α₂KO muscles display deranged energy homeostasis despite enhanced glycolytic flux and PDH_a activity. This may be linked to decreased mitochondrial oxidative capacity.