Abstract: Tumour necrosis factor receptor-associated factor 6 (TRAF6) is a key adaptor protein that plays important role in signalling pathways triggered by the Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) superfamily, which is highly involved in innate immunity. In this study, we investigated expression patterns and preliminary functional analyses of Nile tilapia traf6. In healthy fish, traf6 transcripts were broadly expressed in all examined tissues with the highest expression level in the blood and the lowest in the liver. traf6 was also detected at various embryonic developmental stages. After challenge with S. agalactiae in vivo, upregulated mRNA expression of traf6 was observed in most examined tissues. Moreover, traf6 expression could be significantly induced in Nile tilapia macrophages by treatment with LPS, Poly I: C and S. agalactiae. In addition, overexpression in HEK293T cells showed that TRAF6 was distributed in the cytoplasm and could significantly increase NF-κB activation. Co-immunoprecipitation (Co-IP) assays showed that TRAF6 could interact with IRAK1 (interleukin-1 receptor associated kinase 1), which also plays a vital role in the TLR/IL-1R signalling pathway. In vivo, TLR2, TLR21 and TLR13b overexpression could upregulate traf6 expression levels, which indicates that TRAF6 is involved in the signal transduction of TLR2, TLR21 and TLR13b. These findings suggest that TRAF6 plays important roles in the immune response to pathogen invasion.