Glutathione reductase (Gsr) is an enzyme that reduces glutathione disulfide to the sulfhydryl form, a major cellular antioxidant. Oxidative burst plays an important role in pathogen killing and initiation of inflammation. Proper redox regulation is crucial for balancing effective pathogen elimination and host preservation. We tested the hypothesis that Gsr plays a critical role in host defense by comparing wildtype (WT) and Gsr- mice after E. coli infection. Compared to WT mice, Gsr- mice exhibited substantially higher mortality, associated with greater bacterial burden, cytokine storm, and striking histological abnormalities. Surprisingly, Gsr- mice displayed increased resistance to LPS. While Gsr- mice exhibited defects in phagocyte mobilization, there appeared no substantial defects in either cell signaling or cytokine production in Gsr- macrophages. The role of Gsr in phagocytic oxidative burst was assessed by both flow cytometry and bioluminescence imaging. The oxidative burst in WT neutrophils after PMA stimulation was sustained for more than 30 min, while oxidative burst was very transient in Gsr- cells (<5 min). Likewise, oxidative burst after E. coli infection was also significantly weaker in the Gsr- neutrophils. Thus, Gsr plays a critical role in host defense against bacterial infection by regulating various immune functions, including facilitation of bacterial killing via sustaining oxidative burst in phagocytes, through maintenance of a delicate redox balance.