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. In particular, ROS such as H2O2, produced by NOXs has far postulated to act as a secondary messenger, facilitating insulin signalling by inactivating protein tyrosine phosphatases. The focus of the current study is on the role of skeletal-muscle and liver NOX4 in glucose metabolism, insulin sensitivity and insulin signalling. Muscle-specific NOX4 knockout mice [Mck-Cre; Nox4(fl/fl)] and liver-specific NOX4 knockout mice [Alb-Cre;Nox4(fl/fl)] were fed either a standard chow diet or a high-fat diet and subjected to insulin and glucose tolerance tests as well as hyperinsulinaemic - euglycaemic clamps. Furthermore, we isolated primary myoblasts and hepatocytes from Nox4(fl/fl,), Mck-Cre; Nox4(fl/fl) and Alb-Cre;Nox4(fl/fl) mice to delineate the mechanisms involved. Skeletal muscle NOX4-deficiency resulted in glucose intolerance and insulin resistance in both chow and high fat fed mice. Moreover, NOX4 deficiency in liver exacerbated the development of obesity, hepatic steatosis and insulin resistance in mice fed a high fat diet. NOX4 deficiency in myoblasts or hepatocytes also attenuated insulin signalling as assessed by monitoring the PI3K/AKT signalling. Our results highlight the importance of muscle and liver NOX4-derived ROS in the promotion of insulin signalling and the prevention of insulin resistance. Our findings point towards NOX4-derived ROS being required for glucose metabolism.