The aim of this study was to explore the function and regulatory mechanism of tumor necrosis factor receptor-associated factor 3 (TRAF3) in fish antiviral immune response. In the present study, the transcript encoding TRAF3 (AjTRAF3) was obtained from Japanese eel, Anguilla japonica, by using reverse transcription PCR. The structural characteristics of AjTRAF3 were analyzed using bioinformatics softwares and its expression profile and functional mechanism were investigated by qPCR, luciferase reporter and co-immunoprecipitation assay. The open reading frame of AjTRAF3 was 1 707 bp long, encoding a polypeptide consisting of an N-terminal ring domain, two Zn Finger domains, a helical domain, and a C-terminal TRAF-C (MATH) domain. qPCR analysis revealed a wide tissue distribution of AjTRAF3, with the highest expression in brain, followed by head kidney, and the lowest in heart. The highest induction of AjTRAF3 in response to Poly I:C stimulation was observed at 24 hour post injection (hpi) in spleen, being 15.83 fold higher than control. The highest up-regulation of AjTRAF3 after E. tarda infection was observed in spleen at 24 hpi, being 31.47 fold higher than control. Furthermore, the eukaryotic expression plasmid was constructed to study the function of AjTRAF3 in vitro. Our results revealed increased expression of antiviral related genes and increased luciferase transactivation of the AjIFN2, AjIFN4 and NF-κB promoter in cells overexpressed with AjTRAF3. Further, AjRIG-IN, AjMAVS- or AjIRF3-induced AjIFN2, AjIFN4 and NF-κB promoter activation was significantly enhanced in cells co-transfected with AjTRAF3. In addition, subcellular localization analysis revealed the cytoplasmic distribution of AjTRAF3 and the co-localization of AjTRAF3 with MAVS on mitochondria. Lastly, co-immunoprecipitation assays showed that AjTRAF3 interacts with AjMAVS via the MATH domain, whereas a mutant lacking this domain lost the ability to interact with AjMAVS. Collectively, these data suggested that AjTRAF3 can induce antiviral responses by regulating RIG-I/MAVS-mediated signaling. These findings contribute to understanding of function of AjTRAF3 in antiviral response.