The protein tyrosine kinase Src regulates the synthesis of TLR3-mediated IFN-b via the TBK1-IFN regulatory factor 3 axis. However, the molecular mechanisms regulating Src activity in TLR3 signaling remain unclear. In this study, we report that GSK3b regulates Src phosphorylation via TNFR-associated factor 2 (TRAF2)-mediated Src ubiquitination. GSK3b deficiency in mouse embryonic fibroblasts significantly reduces polyinosinic:polycytidylic acid-induced IFN-b and IFN-stimulated gene expression, which is caused by diminished phosphorylation of Src at tyrosine 416. Src undergoes polyinosinic:polycytidylic acid- dependent lysine 63 chain ubiquitination, and TRAF2 is a direct E3 ligase for Src. Our study reveals novel mechanisms underlying TLR3-mediated antiviral responses mediated via the GSK3b-TRAF2-Src axis.