Trim25 is a member of Tripartite Motif (TRIM) family. Previous studies report that trim25 modulates antiviral activity by activating RIG-I. In this study we explored the four alternative splicing (AS) variants X1-X4 of Japanese flounder trim25. The sequences of the AS variants were highly conserved. Expression levels of trim25 X1-X4 were increased after 12 h of poly I:C treatment in vitro. In vivo expression of X2-X4 in liver, kidney (except X2) and blood was significantly up-regulated in early stages of poly I:C treatment. Subcellular localization analysis showed that Trim25 X1-X4 were distributed in different cellular organelles. The recombinant vector pcDNA3.1-Trim25 X1-X4 were successfully overexpressed in Flounder cells and the samples were collected. Expression patterns of RIG-I pathway genes dhx58, traf6, traf2, nfkbia and il-8 were explored in vitro and in vivo after poly I:C treatment, as well as overexpressed samples. The findings of this study imply that AS variants of trim25 confer antiviral activity in Japanese flounder by modulating innate immune response.