AbstractOur laboratory previously showed lipid hydroperoxides and oxylipin levels are elevated in response to loss of skeletal muscle innervation and are associated with muscle pathologies. To elucidate the pathological impact of lipid hydroperoxides, we overexpressed glutathione peroxidase 4 (GPx4), an enzyme that targets reduction of lipid hydroperoxides in membranes, in adult CuZn superoxide dismutase knockout (Sod1KO) mice that show accelerated muscle atrophy associated with loss of innervation. The gastrocnemius muscle from Sod1KO mice shows reduced mitochondrial respiration and elevated oxidative stress (F2‐isoprostanes and hydroperoxides) compared to wild‐type (WT) mice. Overexpression of GPx4 improved mitochondrial respiration and reduced hydroperoxide generation in Sod1KO mice, but did not attenuate the muscle loss that occurs in Sod1KO mice. In contrast, contractile force generation is reduced in EDL muscle in Sod1KO mice relative to WT mice, and overexpression of GPx4 restored force generation to WT levels in Sod1KO mice. GPx4 overexpression also prevented loss of muscle contractility at the single fibre level in fast‐twitch fibres from Sod1KO mice. Muscle fibres from Sod1KO mice were less sensitive to both depolarization and calcium at the single fibre level and exhibited a reduced activation by S‐glutathionylation. GPx4 overexpression in Sod1KO mice rescued the deficits in both membrane excitability and calcium sensitivity of fast‐twitch muscle fibres. Overexpression of GPx4 also restored the sarco/endoplasmic reticulum Ca2+‐ATPase activity in Sod1KO gastrocnemius muscles. These data suggest that GPx4 plays an important role in preserving excitation–contraction coupling function and Ca2+ homeostasis, and in maintaining muscle and mitochondrial function in oxidative stress‐induced sarcopenia. imageKey points Knockout of CuZn superoxide dismutase (Sod1KO) induces elevated oxidative stress with accelerated muscle atrophy and weakness. Glutathione peroxidase 4 (GPx4) plays a fundamental role in the reduction of lipid hydroperoxides in membranes, and overexpression of GPx4 improves mitochondrial respiration and reduces hydroperoxide generation in Sod1KO mice. Muscle contractile function deficits in Sod1KO mice are alleviated by the overexpression of GPx4. GPx4 overexpression in Sod1KO mice rescues the impaired muscle membrane excitability of fast‐twitch muscle fibres and improves their calcium sensitivity. Sarco/endoplasmic reticulum Ca2+‐ATPase activity in Sod1KO muscles is decreased, and it is restored by the overexpression of GPx4. Our results confirm that GPx4 plays an important role in preserving excitation–contraction coupling function and Ca2+ homeostasis, and maintaining muscle and mitochondrial function in oxidative stress‐induced sarcopenia.