Skeletal muscle constantly produces reactive oxygen species (ROS) and ROS generation is increased during exercise. Both mitochondria and NADPH oxidases (NOXs) have been implicated as sources of ROS in muscle, but there is evidence for NOXs being key drivers of exercise-induced ROS. However, definitive evidence for this and the precise NOX involved remain unknown. Contraction-induced ROS generation is important in driving antioxidant defence and mitochondrial adaptive responses that are key to the health-promoting effects of exercise. The mechanisms by which ROS coordinate antioxidant defence, mitochondrial biogenesis and insulin sensitivity remain incompletely understood. The focus of the current study is on skeletal-muscle NOX4 and its role on skeletal muscle exercise metabolism, antioxidant defence and mitochondrial biogenesis. 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 exercise capacity and endurance tests. Furthermore, primary myoblasts were isolated to delineate the cell intrinsic mechanisms by which NOX4 elicits its effects on mitochondrial biogenesis and antioxidant defence. NOX4-deficiency resulted in reduced muscle mass, energy expenditure and impaired exercise and endurance capacity. In addition, both NOX4-deficient mice and primary myoblasts showed impaired mitochondrial biogenesis and reduced expression of antioxidant defence genes. Our results highlight NOX4 as a key regulator of antioxidant defence, exercise capacity and mitochondrial metabolism.