Tripartite motif (TRIM) proteins play a regulatory function in cancer, cell apoptosis and innate immunity. To understand the role of TRIM39 in Nile tilapia (Oreochromis niloticus), TRIM39 cDNA was isolated. The total length of TRIM39 cDNA was 5025 bp. The deduced OnTRIM39 protein contains 549 amino acids and has conserved domains of the TRIM family, which are the RING, B‐box, coiled‐coil and PRY‐SPRY domains. OnTRIM39 mRNA was widely expressed in various tissues. After challenge with Streptococcus agalactiae and stimulation with polyinosinic polycytidylic acid poly (I:C) and lipopolysaccharides (LPS), the amount of OnTRIM39 transcript was changed in various tested tissues. OnTRIM39 overexpression increased NF‐κB activity. OnTRIM39 was present in the cytoplasm. Mass spectrometry of proteins pulled down with recombinant OnTRIM39 showed that 250 proteins potentially interact with OnTRIM39. The authors selected I3K4I3 from the 250 candidate proteins to verify its interaction with TRIM39. They also selected I3KL45, a member of the same 14‐3‐3 protein family, to verify its interaction with TRIM39. The results of pull‐down assays showed that OnTRIM39 interacted with both I3K413 and I3KL45. These results contribute to further study of the innate immune mechanism of tilapia.