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Francisella tularensis is an intracellular bacterium and as such is expected to encounter a continuous attack by reactive oxygen species (ROS) in its intracellular habitat and efficiently coping with oxidative stress is therefore essential for its survival. The oxidative stress response system of F tularensis is complex and includes multiple antioxidant enzymes and pathways, including the transcriptional regulator OxyR and the H2O2-decomposing enzyme catalase, encoded by katG. The latter is regulated by OxyR. A deletion of either of these genes, however, does not severely compromise the virulence of F tularensis and we hypothesized that if the bacterium would be deficient of both catalase and OxyR, then the oxidative defense and virulence of F tularensis would become severely hampered. To test this hypothesis, we generated a double deletion mutant, Delta oxyR/Delta katG, of F tularensis LVS and compared its phenotype to the parental LVS strain and the corresponding single deletion mutants. In accordance with the hypothesis, Delta oxyR/Delta katG was distinctly more susceptible than Delta oxyR and Delta katG to H2O2, ONOO-, and O-2(-), moreover, it hardly grew in mouse-derived BMDM or in mice, whereas Delta katG and Delta oxyR grew as well as F tularensis LVS in BMDM and exhibited only slight attenuation in mice. Altogether, the results demonstrate the importance of catalase and OxyR for a robust oxidative stress defense system and that they act cooperatively. The lack of both functions render F tularensis severely crippled to handle oxidative stress and also much attenuated for intracellular growth and virulence.