TNF receptor-associated factor 6 (TRAF6) is an essential adaptor protein for the Interleukin-1 (IL-1) signaling pathway; however, its role in the signaling of another proinflammatory cytokine, tumor necrosis factor alpha (TNFalpha, has not been explored. Interestingly, we observed that TNFalpha-induced expression of IL-6, CXCL1 and granulocyte macrophage colony stimulating factor (GM-CSF) were significantly enhanced in TRAF6-deficient MEFs. Compared to those observed in wild-type MEFs, TNFalpha-induced IkappaB kinase (IKK) activation and IkappaBalpha degradation were enhanced in TRAF6-deficient MEFs. Also, TNFalpha-induced DNA binding activity and transcriptional activation of nuclear factor kappaB (NF-kappaB) were also augmented in TRAF6-deficient MEFs. On the other hand, TRAF6 deficiency did not affect the TNFalpha-induced activation of mitogen-activated protein (MAP) kinases, ERK, JNK, and p38. Moreover, the reintroduction of exogenous TRAF6 into TRAF6-deficient MEFs clearly suppressed TNFalpha-induced IKK activation, NF-kappaB activation and subsequent cytokine expression. In contrast, both the deletion mutant (DeltaN) and the point mutant (C70A) of TRAF6, which is defective in its ubiquitin ligase activity, failed to repress TNFalpha-induced IKK activation, NF-kappaB activation and cytokine production. Thus, these data suggest that TRAF6 negatively regulates TNFalpha-induced NF-kappaB activation through its ubiquitin ligase activity.