Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N 2 ) to ammonia (NH 3 ). Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Nitrogen-fixing bacteria rely on the protein CowN to grow in the presence of CO. However, the mechanism by which CowN operates is unknown. Here, we present the biochemical characterization of CowN and examine how CowN protects nitrogenase from CO. We determine that CowN interacts directly with nitrogenase and that CowN protection observes hyperbolic kinetics with respect to CowN concentration. At a CO concentration of 0.001 atm, CowN restores nearly full nitrogenase activity. Our results further indicate that CowN’s protection mechanism involves decreasing the binding affinity of CO to nitrogenase’s active site approximately tenfold without interrupting substrate turnover. Taken together, our work suggests CowN is an important auxiliary protein in nitrogen fixation that engenders CO tolerance to nitrogenase.