The NADPH oxidase 2 (NOX2) complex is responsible for the production of ROS in phagocytic cells. Genetic defects in NOX2 lead to opportunistic infections and inflammatory manifestations such as granulomas in humans, also known as chronic granulomatous disease (CGD). This condition is mirrored in mice with defective ROS production and interestingly both species are predisposed to autoimmune diseases. An unresolved question is whether the hyper‐inflammation and tendency to develop autoimmunity are secondary to the increased infections, or whether these are parallel phenomena. We generated germ‐free ROS deficient Ncf1 mutant mice that when reared in specific pathogen‐free condition, are highly susceptible to collagen‐induced arthritis compared with wild‐type mice. Strikingly, arthritis incidence and severity was almost identical in germ‐free and specific pathogen‐free ROS‐deficient mice. In addition, partial reduction of the microbial flora by antibiotics treatment did not alter the disease course. Taken together, this shows that ROS has a clear immune regulatory function that is decoupled from its function in host defence.