The NAD(P)H:quinone oxidoreductase 1 (NQO1) is a phase II enzyme that reduces and detoxifies quinones and their derivatives. Although overexpressed in tumor cells, the NQO1 has been linked with the suppression of carcinogenesis, and the effect of NQO1 on tumor necrosis factor (TNF), a cytokine that mediates tumorigenesis through proliferation, invasion, angiogenesis, and metastasis of tumors, is currently unknown. The purpose of our study was to determine the role of NQO1 in TNF cell signaling by using keratinocytes derived from wild-type and NQO1 gene-deleted mice. TNF induced nuclear factor (NF)-kappaB activation in wild-type but not in NQO1-deleted cells. The treatment of wild-type cells with dicoumarol, a known inhibitor of NQO1, also abolished TNF-induced NF-kappaB activation. NF-kappaB activation induced by lipopolysaccharide, phorbol ester, and cigarette smoke, was also abolished in NQO1-deleted cells. The suppression of NF-kappaB activation was mediated through the inhibition of IkappaBalpha kinase activation, IkappaBalpha phosphorylation, and IkappaBalpha degradation. Further, the deletion of NQO1 abolished TNF-induced c-Jun N-terminal kinase, Akt, p38, and p44/p42 mitogen-activated protein kinase activation. TNF also induced the expression of various NF-kappaB-regulated gene products involved in cell proliferation, antiapoptosis, and invasion in wild-type NQO1 keratinocytes but not in NQO1-deleted cells. The suppression of these antiapoptotic gene products increased TNF-induced apoptosis in NQO1-deleted cells. We also found that TNF activated NQO1, and NQO1-specific small interfering RNA abolished the TNF-induced NQO1 activity and NF-kappaB activation. Overall, our results indicate that NQO1 plays a pivotal role in signaling activated by TNF and other inflammatory stimuli and that its suppression is a potential therapeutic strategy to inhibit the proliferation, survival, invasion, and metastasis of tumor cells.