Reactive oxygen species, produced by all living organisms as natural by-products of oxygen metabolism and by specialised enzymes known as NAPDH oxidases (NOXs), have been shown to elicit both deleterious and protective effects in various human diseases, including obesity and type 2 diabetes. The focus of the current study is on skeletal-muscle NOX4 and its role on muscle glucose metabolism and mitochondrial function. Muscle-specific NOX4 knockout mice [Mck-Cre;Nox4(fl/fl)] were fed either a standard chow diet or a high-fat diet and were then subjected to insulin and glucose tolerance tests as well as hyperinsulinaemic/euglycaemic clamps. NOX4-deficiency resulted in reduced Nox4 mRNA and NOX4 protein, and was accompanied by glucose intolerance and insulin resistance after high fat feeding. High fat-fed NOX4-deficient mice showed reduced energy expenditure, mitochondrial gene expression and mitochondrial capacity (as assessed by measuring citrate synthase activity) and reduced expression of antioxidant defence genes including Nrf2. These results indicate that muscle-specific NOX4 deficiency promotes insulin resistance, glucose intolerance, as well as impaired mitochondrial function in high fat-fed mice. Our results highlight the importance of muscle NOX4 on muscle glucose and mitochondrial metabolism.