Due to the mass and functions in metabolism, skeletal muscle is one of major organs regulating whole-body metabolic homeostasis. SIRT6, a histone deacetylase, has been shown to regulate metabolism in liver and brain, however, its specific role in skeletal muscle is undetermined. In the present study we explored physiological function of SIRT6 in muscle. We generated a muscle-specific SIRT6 knockout mouse model. The mice with SIRT6 deficiency in muscle displayed impaired glucose homeostasis and insulin sensitivity, attenuated whole-body energy expenditure and weakened exercise performance. Mechanistically, deletion of SIRT6 in muscle decreased expression of genes involved in glucose and lipid uptake, fatty acid oxidation and mitochondrial oxidative phosphorylation in muscle cells due to the reduced AMPK activity. In contrast, overexpression of SIRT6 in C2C12 myotubes activates AMPK. Our results from both gain- and loss-of-function experiments identify SIRT6 as a physiological regulator of muscle mitochondrial function. These findings indicate that SIRT6 is a potential therapeutic target for treatment of T2DM.