The ubiquitously expressed G protein -subunit G_s is required for receptor-stimulated intracellular cAMP responses, and is an important regulator of energy and glucose metabolism. We have generated skeletal muscle-specific G_sKO mice (MGsKO) by mating G_s-floxed mice with muscle creatine kinase-cre transgenic mice. MGsKO mice had normal body weight and composition, and their serum glucose, insulin, free fatty acid and triglyceride levels were similar to that of controls. However, MGsKO mice were glucose intolerant despite the fact that insulin sensitivity and glucose-stimulated insulin secretion were normal, suggesting an insulin-independent mechanism. Isolated muscles from MGsKO mice had increased basal glucose uptake and normal responses to a stimulator of AMP-activated protein kinase (AMPK), which indicates that AMPK and its downstream pathways are intact. Compared with control mice, MGsKO mice had reduced muscle mass with decreased cross-sectional area and force production. In addition, adult MGsKO mice showed an increased proportion of type I (slow-twitch, oxidative) fibers based upon kinetic properties and myosin heavy chain isoforms, despite the fact that these muscles have reduced expression of peroxisome proliferators-activated receptor coactivator protein-1 (PGC-1) and reduced mitochondrial content and oxidative capacity. Therefore G_s deficiency led to fast-to-slow fiber-type switching, which appeared to be dissociated from the expected change in oxidative capacity. MGsKO mice are a valuable model for future studies of the role of G_s signaling pathways in skeletal muscle adaptation and their effects on whole body metabolism.