Although the gastrointestinal tract is regarded as mainly anoxic, low O 2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O 2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O 2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile . This enteropathogen has developed efficient strategies to detoxify O 2 . In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O 2 tolerance in C. difficile . These enzymes are responsible for the reduction of O 2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O 2 -reductases are crucial in the ability of C. difficile to tolerate O 2 and survive to O 2 concentrations encountered in the gastrointestinal tract.