Phagocytes generate nitric oxide (NO) and other reactive oxygen and nitrogen species in large quantities to combat infecting bacteria. Here, we report the surprising observation that in vivo survival of a notorious pathogen-Bacillus anthracis-critically depends on its own NO-synthase (bNOS) activity. Anthrax spores (Sterne strain) deficient in bNOS lose their virulence in an A/J mouse model of systemic infection and exhibit severely compromised survival when germinating within macrophages. The mechanism underlying bNOS-dependent resistance to macrophage killing relies on NO-mediated activation of bacterial catalase and suppression of the damaging Fenton reaction. Our results demonstrate that pathogenic bacteria use their own NO as a key defense against the immune oxidative burst, thereby establishing bNOS as an essential virulence factor. Thus, bNOS represents an attractive antimicrobial target for treatment of anthrax and other infectious diseases.