We examined the role of redox signaling generated by NADPH oxidase in activation of NF-κB and host defense against Pseudomonas aeruginosa pneumonia. Using mice with an NF-κB-driven luciferase reporter construct (HIV-LTR/luciferase (HLL)), we found that intratracheal administration of P. aeruginosa resulted in a dose-dependent neutrophilic influx and activation of NF-κB. To determine the effects of reactive oxygen species generated by the NADPH oxidase system on activation of NF-κB, we crossbred mice deficient in p47 phox with NF-κB reporter mice (p47 phox−/− HLL). These p47 phox−/− HLL mice were unable to activate NF-κB to the same degree as HLL mice with intact NADPH oxidase following P. aeruginosa infection. In addition, lung TNF-α levels were significantly lower in p47 phox−/− HLL mice compared with HLL mice. Bacterial clearance was impaired in p47 phox−/− HLL mice. In vitro studies using bone marrow-derived macrophages showed that Toll-like receptor 4 was necessary for NF-κB activation following treatment with P. aeruginosa. Additional studies with macrophages from p47 phox−/− mice confirmed that redox signaling was necessary for maximal Toll-like receptor 4-dependent NF-κB activation in this model. These data indicate that the NADPH oxidase-dependent respiratory burst stimulated by Pseudomonas infection contributes to host defense by modulating redox-dependent signaling through the NF-κB pathway.