NAD(P)H:quinone oxidoreductase 1 (NQO1) is originally identified as a flavoenzyme that catalyzes the two-electron reduction of quinones to their hydroquinone forms, protecting cells from oxidative stress, redox cycling, and neoplastic lesion. Here we found a novel role of NQO1 in Toll-like receptors (TLRs)-mediated innate immune responses. In macrophages, expression of NQO1 was induced by TLR-stimulation, and NQO1-deficient macrophages showed increased production of IL-6, IL-12 and GM-CSF upon LPS stimulation, whereas production of TNF-a and IL-10 was not changed. Consequently, NQO1-deficient mice exhibited increased susceptibility against LPS-induced septic shock, according to the absence of NQO1 in macrophages. NQO1 interacted with a nuclear IκB protein IκB-ζ, which is essential for TLR-mediated induction of a subset of secondary response genes including IL-6, and promoted degradation of IκB-ζ in ubiquitin-dependent manner. We finally identified PDLIM2 as a ubiquitin E3 ligase for NQO1-dependent IκB-ζ degradation. NQO1 augmented association between PDLIM2 and IκB-ζ, resulting in increased degradation of IκB-ζ. Collectively, we demonstrate a negative-feedback pathway of TLR signaling by the NQO1-PDLIM2 complex, a novel ubiquitin E3 ligase for IκB-ζ.