Key points There is lower fat oxidation during post‐exercise recovery in mice lacking 5′‐AMP activated protein kinase α2 (AMPKα2). AMPKα2 is involved in post‐transcriptional and not transcriptional regulation of pyruvate dehydrogenase kinase 4 (PDK4) in muscle. Exercise‐induced AMPKα2 activity increases PDK4 protein content, in turn inhibiting pyruvate dehydrogenase activity and glucose oxidation. The mechanism for increased post‐exercise fat oxidation is by inhibition of carbohydrate oxidation allowing increased fat oxidation rather than by direct stimulation of fat oxidation. Abstract It is well known that exercise has a major impact on substrate metabolism for many hours after exercise. However, the regulatory mechanisms increasing lipid oxidation and facilitating glycogen resynthesis in the post‐exercise period are unknown. To address this, substrate oxidation was measured after prolonged exercise and during the following 6 h post‐exercise in 5´‐AMP activated protein kinase (AMPK) α2 and α1 knock‐out (KO) and wild‐type (WT) mice with free access to food. Substrate oxidation was similar during exercise at the same relative intensity between genotypes. During post‐exercise recovery, a lower lipid oxidation (P < 0.05) and higher glucose oxidation were observed in AMPKα2 KO (respiratory exchange ratio (RER) = 0.84 ± 0.02) than in WT and AMPKα1 KO (average RER = 0.80 ± 0.01) without genotype differences in muscle malonyl‐CoA or free‐carnitine concentrations. A similar increase in muscle pyruvate dehydrogenase kinase 4 (PDK4) mRNA expression in WT and AMPKα2 KO was observed following exercise, which is consistent with AMPKα2 deficiency not affecting the exercise‐induced activation of the PDK4 transcriptional regulators HDAC4 and SIRT1. Interestingly, PDK4 protein content increased (63%, P < 0.001) in WT but remained unchanged in AMPKα2 KO. In accordance with the lack of increase in PDK4 protein content, lower (P < 0.01) inhibitory pyruvate dehydrogenase (PDH)‐E1α Ser293 phosphorylation was observed in AMPKα2 KO muscle compared to WT. These findings indicate that AMPKα2 regulates muscle metabolism post‐exercise through inhibition of the PDH complex and hence glucose oxidation, subsequently creating conditions for increased fatty acid oxidation.