Several studies have shown a substantial increase in fatty acid (FA) oxidation in the post-exercise period compared to the resting state. Even with an increased dietary intake of carbohydrates post-exercise, where a high carbohydrate oxidation would be expected, the contribution of FA for oxidation is high. It appears that muscle glycogen resynthesis after exercise has such a high metabolic priority that utilisation of lipids is elevated to cover the energy expenditure in muscle cells. The mechanisms regulating the selection of energy substrate towards oxidation/resynthesis in muscle during recovery from exercise remain unsolved. To investigate whether 5’AMP activated protein kinase (AMPK) plays a role in this regulation, we measured substrate oxidation in metabolic chambers in AMPKα2 knock-out (KO) and wild type (WT) mice during six hours, following a prolonged exercise bout, in which period the mice had free access to food and water. While relative substrate oxidation was similar during exercise performed at the same relative intensity between genotypes, the AMPKα2 KO during post exercise recovery displayed a lower (p<0.05) FA oxidation (respiratory exchange ratio (RER)=0.84±0.02) than WT mice (RER=0.79±0.01). This could not be explained by the AMPK-ACC-malonyl-CoA axis in the muscle. Instead, data revealed a role of AMPK in the regulation of the PDH complex in the skeletal muscle, which can explain the difference in RER between AMPKα2 KO and WT mice during post exercise recovery. Thus, while AMPKα2 seems not to be of importance for regulation of lipid utilisation in skeletal muscle during exercise, AMPKα2 seems to play an important role for substrate selection during post exercise recovery by inhibiting the activity of PDH complex, in turn increasing lipid oxidation and thus directing carbohydrates to muscle glycogen resynthesis.