TRAF6 is critical for the production of inflammatory cytokines in various TLR-mediated signalling pathways. However, it is poorly understood how TRAF6 regulates TLR3 responses. Here we demonstrate that GSK3 beta interacts with TRAF6 and positively regulates the TLR3-mediated signalling. Suppression of GSK3 beta expression or its kinase activity drastically reduces the production of inflammatory cytokines and the induction of c-Fos by decreasing ERK and p38 phosphorylation. GSK3 beta physically associates with TRAF6 in a TLR3 ligand poly I:C-dependent manner. TRAF6 is determined to be a direct E3 ligase for GSK3 beta, and TRAF6-mediated GSK3 beta ubiquitination is essential for poly I:C-dependent cytokine production by promoting the TLR3 adaptor protein TRIF-assembled signalling complex.