Abstract Virus infection induces the MAVS–TNFR-associated factor (TRAF) 3 signaling axis on mitochondria. It remains to elucidate the corresponding regulatory processes. In this study, we identify UXT-V1 as a novel TRAF3-binding protein. UXT-V1 is critical for the virus-induced activation of NF-κB and IFN regulatory factor 3. Reduction of UXT-V1 impairs the induction of IFN-β and attenuates the host antiviral responses. The N-terminal TRAF-binding motif of UXT-V1 binds to the C-terminal TRAF domain of TRAF3, thus facilitating the interaction between TRAF3 and MAVS. Notably, TRAF3 and TNFR-associated death domain protein are recruited onto mitochondria upon virus infection. These translocations are blocked when knocking down UXT-V1. Thus, UXT-V1 represents a novel integral component of the MAVS signalosome on mitochondria, mediating the innate antiviral signal transduction.