Hexose-6-phosphate dehydrogenase (H6PD) is the initial component of a pentose phosphate pathway inside the endoplasmic reticulum (ER) that generates NADPH for ER enzymes. In liver H6PD is required for the 11-oxoreductase activity of 11beta-hydroxysteroid dehydrogenase type 1, which converts inactive 11-oxo-glucocorticoids to their active 11-hydroxyl counterparts; consequently, H6PD null mice are relatively insensitive to glucocorticoids, exhibiting fasting hypoglycemia, increased insulin sensitivity despite elevated circulating levels of corticosterone, and increased basal and insulin-stimulated glucose uptake in muscles normally enriched in type II (fast) fibers, which have increased glycogen content. Here, we show that H6PD null mice develop a severe skeletal myopathy characterized by switching of type II to type I (slow) fibers. Running wheel activity and electrically stimulated force generation in isolated skeletal muscle are both markedly reduced. Affected muscles have normal sarcomeric structure at the electron microscopy level but contain large intrafibrillar membranous vacuoles and abnormal triads indicative of defects in structure and function of the sarcoplasmic reticulum (SR). SR proteins involved in calcium metabolism, including the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), calreticulin, and calsequestrin, show dysregulated expression. Microarray analysis and real-time PCR demonstrate overexpression of genes encoding proteins in the unfolded protein response pathway. We propose that the absence of H6PD induces a progressive myopathy by altering the SR redox state, thereby impairing protein folding and activating the unfolded protein response pathway. These studies thus define a novel metabolic pathway that links ER stress to skeletal muscle integrity and function.