The human DEAD-box helicase 3 (DDX3) has been shown to contribute to type I interferon induction downstream of anti-viral pattern recognition receptors (PRRs). It binds to TANK-binding kinase 1 (TBK1) and IB-kinase- (IKK, the two key kinases mediating activation of Interferon regulatory factor (IRF) 3 and IRF7. We previously demonstrated that DDX3 facilitates IKK activation downstream of RIG-I and then links the activated kinase to IRF3. In this study, we probed the interactions between DDX3 and other key signalling molecules in the RIG-I pathway and identified a novel direct interaction between DDX3 and TRAF3 mediated by a TRAF-interaction motif in the N-terminus of DDX3, which was required for TRAF3 ubiquitination. Interestingly, we observed two waves of K63-linked TRAF3 ubiquitination following RIG-I activation by Sendai Virus infection (SeV), both of which were suppressed by DDX3 knockdown. We also investigated the spatiotemporal formation of endogenous downstream signalling complexes containing the MAVS adaptor, DDX3, IKK, TRAF3 and IRF3. DDX3 was recruited to MAVS early after SeV infection, suggesting it might mediate subsequent recruitment of other molecules. Indeed, knockdown of DDX3 prevented formation of TRAF3-MAVS and TRAF3-IKKcomplexes. Based on our data, we propose that early TRAF3 ubiquitination is required for formation of a stable MAVS-TRAF3 complex, while the second wave of TRAF3 ubiquitination mediates IRF3 recruitment and activation. Our study characterises DDX3 as a multifunctional adaptor molecule that coordinates assembly of different TRAF3, IKK and IRF3-containing signalling complexes downstream of MAVS. Additionally, it provides novel insights into the role of TRAF3 in RIG-I signalling.