Excess reactive oxygen species (ROS) induced by physical inactivity is associated with muscle atrophy and muscle weakness. However, the role of mitochondrial ROS on disuse-induced muscle atrophy is not fully understood. The purpose of this study was to utilize a genetic strategy to examine the effect of neutralizing mitochondrial ROS on disuse-induced skeletal muscle atrophy. This was accomplished by placing wild-type (WT) and mitochondrial-targeted catalase-expressing (MCAT) littermate mice on 7 days of hindlimb unloading. After assessment of body weight and composition, muscles were analyzed for individual muscle mass, force-generating capacity, fiber type, cross-sectional area, and mitochondrial function, including H2O2 production. Despite a successful attenuation of mitochondrial ROS, MCAT mice were not protected from muscle atrophy. No differences were observed in body composition, lean mass, individual muscle masses, force-generating capacity, or muscle fiber cross-sectional area. These data suggest that neutralizing mitochondrial ROS is insufficient to suppress disuse-induced loss of skeletal muscle mass and contractile function.NEW & NOTEWORTHY The premise of this study was to examine the efficacy of genetic suppression of mitochondrial reactive oxygen species (ROS) to attenuate disuse-induced muscle atrophy and muscle weakness. Neutralization of mitochondrial ROS by MCAT expression was insufficient to rescue muscle atrophy and muscle weakness.