Background & Aims: Exercise as a lifestyle modification is frontline therapy for NAFLD, but how components of exercise attenuate steatosis is unclear. Approach & Results: To uncouple the effect of increased muscle mass from weight loss in obesity, Myostatin knockout mice were bred on a lean and obese db/db background. Myostatin deletion increases gastrocnemius mass, reduces hepatic steatosis and hepatic Srebp1 expression in obese mice, with no impact on adiposity or body weight. Interestingly, hypermuscularity reduces hepatic NADPH oxidase 1 (Nox1) expression, but not Nox4, in db/db mice. To evaluate a deterministic function of Nox1 on steatosis, Nox1 knockout mice were bred on a lean and db/db background. NOX1 deletion significantly attenuates hepatic oxidant stress, steatosis, and Srebp1 programming in obese mice to parallel hypermuscularity, with no improvement in adiposity, glucose control, or hypertriglyceridemia to suggest off-target effects. Directly assessing the role of NOX1 on SREBP1, insulin-mediated SREBP1 expression was significantly increased in either NOX1, NOXO1, and NOXA1 or NOX5-transfected HepG2 cells vs β-galactosidase control virus, indicating superoxide is the key mechanistic agent for the actions of NOX1 on SREBP1. Metabolic Nox1 regulators were evaluated using physiological, genetic, and diet-induced animal models that modulated upstream glucose and insulin signaling, identifying hyperinsulinemia as the key metabolic derangement explaining Nox1-induced steatosis in obesity. GEO data revealed hepatic NOX1 predicts steatosis in obese humans with biopsy-proven NAFLD. Conclusion: Taken together, these data suggest hypermuscularity attenuates Srebp1 expression in db/db mice through a NOX1-dependent mechanism.