Adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) is a major cellular energy sensor and plays an important role in the regulation of metabolic homeostasis. In order to study the role of the catalytic α2 subunit of AMPK in skeletal muscle energy metabolism, skeletal muscle satellite cells were isolated from the hind legs of AMPKα2+/+ and AMPKα2-/- mice to establish myotube cultures. Radiolabelled glucose and oleic acid were used to measure substrate uptake and oxidation, qPCR analysis for mRNA expression of selected genes and quantitative proteomics was performed to get a global overview of the protein level changes of the cells. In the present work, we showed that myotubes established from AMPKα2-/- mice had lower basal oleic acid oxidation, but exhibited an increased response to mitochondrial uncoupling and no suppression of oleic acid oxidation by glucose. Myotubes from AMPKα2-/- mice also showed lower uptake and oxidation of glucose as well as extinguished response to mitochondrial uncoupling for glucose oxidation compared to myotubes established from AMPKα2+/+ mice. Incorporation of acetate into cellular lipids was lower in myotubes from AMPKα2-/- mice compared to myotubes from AMPKα2+/+ mice. Proteomics analysis revealed that AMPKα2-/- myotubes had upregulated pathways related to mitochondrial function and fatty acid oxidation, and decreased pathways related to cholesterol and fatty acid biosynthesis. In conclusion, ablation of the AMPKα2 catalytic subunit in skeletal muscle cells resulted in increased response to mitochondrial uncoupling for oleic acid oxidation and reduced glucose suppression of oleic acid oxidation, as well as upregulated pathways related to mitochondrial function and fatty acid oxidation and reduced lipid synthesis.