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Interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 are critical signalling mediators and play pivotal roles in the innate immune and inflammatory responses mediated by TLR/IL-1R. In the present study, two IRAK family members, On IRAK1 and On IRAK4, were identified in the Nile tilapia Oreochromis niloticus with a conserved N-terminal death domain and a protein kinase domain, similar to those of other fishes and mammals. The gene structures of On IRAK1 and On IRAK4 are organized into fifteen exons split by fourteen introns and ten exons split by nine introns. On IRAK1 and On IRAK4 were broadly expressed in all of the tissues tested, with the highest expression levels being observed in the blood and the lowest expression levels being observed in the liver. Both genes could be detected from 2 d post-fertilization (dpf) to 8 dpf during embryonic development. Moreover, the expression levels of On IRAK1 and On IRAK4 were clearly altered in all five tissues after Streptococcus agalactiae infection in vivo and could be induced by LPS, Poly I: C, S. agalactiae WC1535 and △CPS in Nile tilapia macrophages. The overexpression of On IRAK1 and On IRAK4 in 293T cells showed that they were both distributed in the cytoplasm and could significantly increase NF-κB activation. Interestingly, after transfection, On IRAK1 significantly upregulated On Myd88-induced NF-κB activation, while On IRAK4 had no effect on On Myd88-induced NF-κB activation. Co-immunoprecipitation (Co-IP) assays showed that On Myd88 did not interact with either On IRAK1 or On IRAK4 and that On IRAK1 did not interact with On IRAK4. Taken together, these findings suggest that On IRAK1 and On IRAK4 could play important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion. • On IRAK1 and On IRAK4 gene were identified in Nile tilapia and were upregulated after S. agalactiae challenge. • On IRAK1 and On IRAK4 induced by LPS, Poly I: C, wild and mutant type S. agalactiae in in vitro. • On Myd88 did not interact with either On IRAK1 or On IRAK4, and On IRAK1 did not interact with On IRAK4. • Overexpression of On IRAK1 and On IRAK4 significantly increased NF-κB activation. • On IRAK1 significantly upregulated the NF-κB activity induced by On Myd88. [ABSTRACT FROM AUTHOR]