TNF receptor-associated factor 6 (TRAF6) plays a key role in the regulation of innate immune responses by mediating signals from both TNF receptors (TNFRs) and interleukin-1 receptors (IL-1Rs)/Toll-like receptors (TLRs). Here, we define a new role for TRAF6 in antagonizing cell death during TNF signaling. In TRAF6-deficient 3T3 (T6 -/- 3T3) cells, TNF stimulation leads to the accumulation of reactive oxygen species (ROS), which in turn results in prolonged c-Jun N-terminal kinase (JNK) activation and accelerated cell death. Furthermore, TNF-induced p65/RelA phosphorylation as well as transcriptional activity of nuclear factor-κB (NF-κB) was significantly downregulated in T6 -/- 3T3 cells. Interestingly, TRAF6 deficiency leads to constitutive phosphorylation and inactivation of glycogen synthase kinase 3β (GSK3β). Restoration of GSK3β activity through exogenous expression of a GSK3β constitutive active form rescued cell death in TRAF6-null 3T3 cells. These data suggest a role for TRAF6 in the maintenance of cell survival by regulating GSK3β activity in TNF signaling.