TANK-BindingKinase 1 (TBK1) is a serine/threonine kinase ubiquitously expressed in many tissues and plays an important role in the RLR antiviral signaling pathway. Upon viral infection, various virus components, including viral DNA, RNA are sensed by different pattern recognition receptors (PRRs). Among these PRRs, TLR3, RIG-I/MDA5, and cGAS recruit different adaptors including TRIF, VISA/ MAVS, and MITA/STING to activate TBK1. Activated TBK1 then promote the phosphorylation and homodimerization of IFN-regulatory factors 3 and 7 (IRF3/7), in turn promoting type-I Interferon (IFN-I) production. In this study, we reported that ZW10 Interacting Kinetochore Protein (ZWINT) positively regulates RLR antiviral signaling by targeting RIG-I/VISA/TBK1. Ectopic expression of ZWINT potentiated Sendai virus-induced, as well as TBK1-or VISA/MAVS-mediated IFN-β promoter activation by enhancing dimerization of IRF3. In contrast, knocking down of ZWINT expression with siRNA resulted in reduced IFN-β promoter activation and IRF3 dimerization induced by Sendai virus, indicating that ZWINT positively regulates TBK1-mediated RLR antiviral signaling.