Abstract: Tripartite motif-containing protein 25 (TRIM25) belongs to the E3 ubiquitin ligase family and plays an important role in innate immune response. To study the role of TRIM25 gene in innate antiviral immune response of large yellow croaker (Larimichthys crocea), the TRIM25 gene (named LcTRIM25) of large yellow croaker was cloned and identified. The coding sequence of LcTRIM25 gene is 2097 bp (GenBank accession numbers: MK327541), encoding 698 amino acid residues. Protein domain analysis found that LcTRIM25 contains a conserved Really Interesting New Gene (RING) domain, a B-box2 domain, a Coiled-coil domain and a variable C-terminal PRY/SPRY domain. Multiple sequence alignment and phylogenetic tree analysis showed that the LcTRIM25 gene had high homology with the Epinephelus coioides, but low homology with mammals, reptiles, amphibians and birds, indicating that the different species were under different selection pressure from environments to reach different evolutionary degrees. The results of real-time fluorescence quantitative PCR revealed that the LcTRIM25 gene was ubiquitously expressed in 9 tested issues of healthy large yellow croaker, with the highest expressions in liver and the lowest expression in heart. The expression of LcTRIM25 gene in peripheral blood, head kidney, spleen and liver were rapidly and significantly upregulated after poly (I:C) stimulation, and then decreased in all tissues. The highest expression level of LcTRIM25 gene was found at 6h in head kidney and spleen, at 12h in liver, and at 24h in peripheral blood, demonstrating that the TRIM25 gene might play a key role in the antiviral immune response of large yellow croaker. This study provides a theoretical basis for further understanding the antiviral immune mechanism of large yellow croaker.