Sepsis syndrome is characterized by a dysregulated inflammatory response to infection. NADPH oxidase-dependent reactive oxygen species (ROS) play significant roles in the pathophysiology of sepsis. We previously showed that disruption of Nrf2, a master regulator of antioxidant defenses, caused a dysregulation of innate immune response that resulted in greater mortality in a polymicrobial sepsis and LPS shock model; however, the underlying mechanisms are unclear. In the current study, compared with wild-type (Nrf2+/+) macrophages, we observed greater protein kinase C-induced NADPH oxidase-dependent ROS generation in Nrf2-disrupted (Nrf2−/−) macrophages that was modulated by glutathione levels. To address the NADPH oxidase-mediated hyperinflammatory response and sepsis-induced lung injury and mortality in Nrf2−/− mice, we used double knockout mice lacking Nrf2 and NADPH oxidase subunit, gp91 phox (Nrf2−/−//gp91 phox−/−). Compared with Nrf2+/+ macrophages, LPS induced greater activation of TLR4 as evident by TLR4 surface trafficking and downstream recruitment of MyD88 and Toll/IL-1R domain-containing adaptor in Nrf2−/− macrophages that was diminished by ablation of gp91 phox. Similarly, phosphorylation of IκB and IFN regulatory factor 3 as well as cytokine expression was markedly higher in Nrf2−/− macrophages; whereas, it was similar in Nrf2+/+ and Nrf2−/−//gp91 phox−/−. In vivo studies showed greater LPS-induced pulmonary inflammation in Nrf2−/− mice that was significantly reduced by ablation of gp91 phox. Furthermore, LPS shock and polymicrobial sepsis induced early and greater mortality in Nrf2−/− mice; whereas, Nrf2−/−//gp91 phox−/− showed prolong survival. Together, these results demonstrate that Nrf2 is essential for the regulation of NADPH oxidase-dependent ROS-mediated TLR4 activation and lethal innate immune response in sepsis.