We have previously shown that double-stranded RNA-triggered, Toll-like receptor 3 (TLR3)-mediated signaling is independent of MyD88, IRAK4, and IRAK. Instead, TRAF6, TAK1, and TAB2 are recruited to TLR3 on poly(I·C) stimulation. TRAF6-TAK1-TAB2 are then translocated to the cytosol where TAK1 is phosphorylated and activated, leading to the activation of IκB kinase and NFκB. The present study addressed two important questions: (i) How are TRAF6, TAK1, and TAB2 recruited to TLR3? (ii) Are TRAF6, TAK1, and TAB2 also required for TLR3-mediated IRF3 activation? Recently, a novel Toll-IL-1 receptor (TIR)-containing adapter, TIR domain-containing adapter inducing IFN-β (TRIF), was shown to play a critical role in TLR3-mediated activation of NF-κB and IRF3. We found that TLR3 recruits TRAF6 via adapter TRIF through a TRAF6-binding sequence in TRIF (PEEMSW, amino acids 250-255). Mutation of this TRAF6-binding sequence abolished the interaction of TRIF with TRAF6, but not with TLR3. Interestingly, mutation of the TRAF6-binding site of TRIF only abolished its ability to activate NF-κB but not IRF3, suggesting that TLR3-mediated activation of NF-κB and IRF3 might bifurcate at TRIF. In support of this finding, we showed that DN-TRAF6 and DN-TAK1 blocked poly(I·C)-induced NF-κB but not IRF3 activation. Furthermore, whereas poly(I·C)-induced NF-κB activation is completely abolished inTRAF6-/- MEFs, the signal-induced activation of IRF3 is TRAF6 independent. In conclusion, TRIF recruits TRAF6-TAK1-TAB2 to TLR3 through its TRAF6-binding site, which is required for NF-κB but not IRF3 activation. Therefore, double-stranded RNA-induced TLR3/TRIF-mediated NF-κB and IRF3 activation diverge at TRIF.