Tumour necrosis factor receptor associated factor 3 (TRAF3) is a crucial transducing protein for linking upstream receptor signals and downstream antiviral signalling pathways. Previous studies mostly clarified the functions of TRAF3 in mammals, birds and fish, but little is known about the characterization and function of TRAF3 in amphibians. In this study, the molecular and functional identification of two TRAF3 genes, AdTRAF3A and AdTRAF3B, were investigated in the Chinese giant salamander Andrias davidianus. The complete open reading frames (ORFs) of AdTRAF3A and AdTRAF3B were 1698 bp and 1743 bp in length, encoding 565 and 580 amino acids, respectively. Both AdTRAF3A and AdTRAF3B deduced proteins contained a RING finger, two TRAF-type zinc fingers, a coiled-coil and a MATH domain. Phylogenetic analysis showed that the AdTRAF3 protein clustered together with other known TRAF3 proteins. Gene expression analysis showed that AdTRAF3s were broadly distributed in all examined tissues with similar distribution patterns. AdTRAF3s in the blood or spleen positively responded to Giant salamander iridovirus (GSIV) and poly (I:C) induction but exhibited distinct response patterns. Silencing AdTRAF3A/B remarkably suppressed the expression of IFN signalling pathway-related genes when leukocytes were treated with DNA virus and the viral RNA analogue. Moreover, overexpression of AdTRAF3A may induce the activation of the IFN-β promoter, and the zinc finger, coiled coil and MATH domains of AdTRAF3A were essential for IFN-β promoter activation. However, the overexpression of AdTRAF3B significantly suppressed IFN-β promoter activity, and its inhibitory effect was enhanced when the RING finger or MATH domain was deleted. Furthermore, AdTRAF3A rather than AdTRAF3B significantly induced NF-κB activation, implying that AdTRAF3A may function as an enhancer in both the IFN and NF-κB signalling pathways. Taken together, our results suggest that the two TRAF3 genes play different crucial regulatory roles in innate antiviral immunity in Chinese giant salamanders.