MicroRNAs have emerged as important regulators of glucose and lipid metabolism in several tissues; however, their role in skeletal muscle remains poorly characterized. We determined the effects of the miR-29 family on glucose metabolism, lipid metabolism, and insulin responsiveness in skeletal muscle. We provide evidence that miR-29a and miR-29c are increased in skeletal muscle from patients with type 2 diabetes and are decreased following endurance training in healthy young men and in rats. In primary human skeletal muscle cells, inhibition and overexpression strategies demonstrate that miR-29a and miR-29c regulate glucose uptake and insulin-stimulated glucose metabolism. We identified that miR-29 overexpression attenuates insulin signaling and expression of insulin receptor substrate 1 and phosphoinositide 3-kinase. Moreover, miR-29 overexpression reduces hexokinase 2 expression and activity. Conversely, overexpression of miR-29 by electroporation of mouse tibialis anterior muscle decreased glucose uptake and glycogen content in vivo, concomitant with decreased abundance of GLUT4. We also provide evidence that fatty acid oxidation is negatively regulated by miR-29 overexpression, potentially through the regulation of peroxisome proliferator–activated receptor γ coactivator-1α expression. Collectively, we reveal that miR-29 acts as an important regulator of insulin-stimulated glucose metabolism and lipid oxidation, with relevance to human physiology and type 2 diabetes.