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Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a multifunctional adaptor protein in innate and acquired immune system that plays a key role in the regulation of the RIG-I-like receptor (RLR) and Toll-like receptor (TLR) signaling pathway in mammals. However, the immune function of TRAF3 homologs in freshwater mollusks is not well understood. In this study, we identified a bivalve TRAF3 gene (AwTRAF3) from Anodonta woodiana and investigated its potential roles during immune challenges. The present AwTRAF3 encoded a polypeptide of 562 amino acids with predicted molecular mass of 64.5 kDa and PI of 7.9. Similar to other reported TRAF3s, AwTRAF3 contained a RING finger domain, two TRAF domains with zinc finger domains, a coiled coli region and a conserved C-terminal meprin and TRAF homology (MATH) domain. Quantitative real-time PCR (qRT-PCR) analysis revealed that AwTRAF3 mRNA was broadly expressed in all of the examined tissues, with high expression in hepatopancreas, gill and heart. In addition, immune challenge experiments directly showed that transcript levels of AwTRAF3 in hepatopancreas were significantly regulated upon bacterial (Vibrio alginolyticus and Staphylococcus aureus) and viral (poly (I:C)) challenges, respectively. Moreover, GFP-tagged AwTRAF3 fusion protein was found to be located primarily in the cytoplasm in HEK293T cells. Altogether, these data provided the first experimental demonstration that freshwater mollusks possess a functional TRAF3 that was involved in the innate defense against bacterial and viral infection.The first experimental evidence for presence of TRAF3 in freshwater mollusk.AwTRAF3 displayed structural characteristics typical of TRAF3 family proteins.AwTRAF3 was ubiquitously expressed in various adult tissues of Anodonta woodiana.AwTRAF3 mRNA level was significantly regulated by bacterial and viral challenges.AwTRAF3 was a cytoplasm localization protein in HEK293 cell line.