Abstract: Interferon regulatory factor 3 and 7 (IRF3 and IRF7), which serve as key transcription factors in IFN activation, are tightly regulated to fulfill normal physiological functions. In this study, we found that SMYD3 (SET and MYND domain containing 3), a lysine methylase, exerts a negative regulatory effect on antiviral innate immunity in Siniperca chuatsi. Firstly, amino acid sequence alignment, protein structure prediction, and phylogenetic analysis indicated that the SMYD3 gene in Siniperca chuatsi exhibits high evolutionary conservation. Furthermore, overexpression of Sc-SMYD3significantly suppressed antiviral gene expression upon Siniperca chuatsi Rhabdovirus (SCRV) infection. Mechanistically, SMYD3 interacts with the key transcription factors IRF3 and IRF7 and inhibits the activity to activate interferon promoter and interferon-sensitive response element (ISRE) reporters. Finally, using zebrafish as a model, we found that smyd3-deficient zebrafish exhibited enhanced resistance to SCRV infection compared to wild-type zebrafish. Our results reveal a novel function of the lysine methyltransferase SMYD3 during SCRV infection and identify SMYD3 as a potential target for breeding new Siniperca chuatsi strains with anti-SCRV ability.