Intracellular pathogens must maintain redox homeostasis against the antimicrobial actions of reactive oxygen and nitrogen species produced by host cells. This study proves that glutathione is required to promote survival of an enteric pathogen Salmonella under the conditions producing reactive oxygen or nitrogen species. Glutathione is the non-protein thiol compound distributed in a variety of organisms and possesses strong electron-donating capability to reduce intracellular redox environment. To examine the role of glutathione on Salmonella redox homeostasis under oxidative and nitrosative stress conditions, gshB gene encoding glutathione synthetase was mutated by the one-step PCR inactivation method. The growth of gshB mutant Salmonella producing virtually no glutathione was greatly impaired in the culture media containing either hydrogen peroxide or nitric oxide donors. The results suggest that physiological levels of glutathione can provide a fundamental capability to maintain redox homeostasis for Salmonella in surviving oxidizing conditions of host cells.