The metabolic property of skeletal muscle is highly malleable and adapts to various physiological demands by shifting energy-substrate metabolism. Skeletal muscle metabolism has a significant impact on whole-body metabolism and substrate utilization. Glucose and lipids are the main oxidative fuel substrates in skeletal muscle, and their utilization is coordinated by complex regulatory mechanisms. In people with Type 2 diabetes, glucose uptake and lipid oxidation in skeletal muscle are impaired. These metabolic defects are coupled to impaired insulin signaling. Exercise increases glucose uptake and lipid oxidation by an insulin-independent mechanism. The AMP-activated protein kinase (AMPK) cascade is activated in response to metabolic stress and has therefore been implicated in the regulation of exercise-induced metabolic and gene regulatory responses. AMPK is a heterotrimeric complex composed of a catalytic α, and regulatory β and γ subunits. Selective regulation of AMPK in skeletal muscle may be achieved by targeting α1/β2/γ3 heterotrimeric complexes. Activation of AMPK enhances GLUT4 translocation of glucose uptake in skeletal muscle from Type 2 diabetic patients and animal models of the disease by an insulin-independent mechanism. Transgenic overexpression of mutated forms of the AMPK γ3 subunit provide evidence that activation of AMPK promotes lipid oxidation and prevents the development of skeletal muscle insulin resistance. Thus, AMPK provides a molecular entry point into novel regulatory pathways to enhance lipid and glucose metabolism in an effort to prevent and treat skeletal muscle insulin resistance associated with Type 2 diabetes.