5′-AMP–activated protein kinase (AMPK) is important for metabolic sensing. We used AMPKγ3 mutant–overexpressing Tg-Prkag3^225Q and AMPKγ3-knockout Prkag3^−/− mice to determine the role of the AMPKγ3 isoform in exercise-induced metabolic and gene regulatory responses in skeletal muscle. Mice were studied after 2 h swimming or 2.5 h recovery. Exercise increased basal and insulin-stimulated glucose transport, with similar responses among genotypes. In Tg-Prkag3^225Q mice, acetyl-CoA carboxylase (ACC) phosphorylation was increased and triglyceride content was reduced after exercise, suggesting that this mutation promotes greater reliance on lipid oxidation. In contrast, ACC phosphorylation and triglyceride content was similar between wild-type and Prkag3^−/− mice. Expression of genes involved in lipid and glucose metabolism was altered by genetic modification of AMPKγ3. Expression of lipoprotein lipase 1, carnitine palmitoyl transferase 1b, and 3-hydroxyacyl–CoA dehydrogenase was increased in Tg-Prkag3^225Q mice, with opposing effects in Prkag3^−/− mice after exercise. GLUT4, hexokinase II (HKII), and glycogen synthase mRNA expression was increased in Tg-Prkag3^225Q mice after exercise. GLUT4 and HKII mRNA expression was increased in wild-type mice and blunted in Prkag3^−/− mice after recovery. In conclusion, the Prkag3^225Q mutation, rather than presence of a functional AMPKγ3 isoform, directly promotes metabolic and gene regulatory responses along lipid oxidative pathways in skeletal muscle after endurance exercise.