AMP-activated protein kinase (AMPK) is a conserved, ubiquitously expressed eukaryotic enzyme that is activated in response to increasing AMP level. Regulation of AMPK activity in skeletal muscle is coordinated by contraction and phosphorylation by upstream kinases and a growing number of hormones and cytokines. Once activated, AMPK turns on catabolic, ATP-generating pathways, and turns off ATP-consuming metabolic processes such as biosynthesis and proliferation. Activation of AMPK promotes glucose uptake and fatty acid oxidation, and enhances glycogen storage capacity in skeletal muscle. Interestingly, increased glucose uptake in response to AMPK activation may occur in an insulin-independent manner. It has been confirmed that AMPK is an indirect molecular target of the antidiabetic drug metformin, and it is postulated that AMPK may be responsible for health benefits of exercise. Understanding of AMPK involving molecular pathways that govern skeletal muscle metabolism is of special interest and offers a unique possibility to find new physiological and/or pharmacological strategies that can improve insulin sensitivity. Here we review present knowledge on the physiological function of AMPK in muscle, and highlight its potential role in glucose homeostasis regulation.