Expression of Esr1/ESR1 (encodes ERa) in metabolic tissues is inversely associated with the insulin resistance index HOMA-IR and adiposity in mice and humans and is reduced in muscle from women displaying clinical features of the metabolic syndrome. Because skeletal muscle is responsible for a large proportion of insulin-stimulated glucose disposal, we determined the role of ERa in regulating skeletal muscle metabolism and insulin action by generating mice with muscle-specific knockout (MERKO) or overexpression (mERaOE) of Esr1. Male and female MERKO mice develop insulin resistance with aging, and skeletal muscle from these animals is distinguished by reduced mitochondrial DNA replication and mitochondrial fission signaling concomitant with impaired fatty acid oxidation. Moreover, muscular endurance was reduced, and mitochondrial adaptations to exercise training were blunted in muscle from animals lacking Esr1 in skeletal muscle. In contrast, mERaOE mice showed enhanced skeletal muscle insulin sensitivity, increased mtDNA copy number, and protection against diet-induced insulin resistance that was underscored by improvements muscle mitochondrial fission dynamics and oxidative capacity. Volitional wheel running was increased in mERaOE mice, and muscle from exercise trained mERaOE mice showed significant increases in mtDNA copy number and oxidative capacity over controls. In contrast to the elongated and hyperfused mitochondrial phenotype of MERKO muscle, mitochondria from mERaOE animals were smaller and more spherical with markedly increased cristae density compared with controls. These data are consistent with prior observations indicating strong links between mitochondrial form and metabolic function. Collectively our findings suggest that ERa is critical in the control of mitochondrial membrane architecture and substrate metabolism, and that muscle ESR1 may be an effective therapeutic target to combat metabolic-related diseases in women and men.