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Human DEAD box ATP-dependent RNA helicase DDX3X has been demonstrated to exert crucial functions in carcinogenesis and antiviral immune response. However, to our knowledge, few information focused on the functions of fish DDX3X. In this study, we cloned and characterized a DDX3X homolog from orange spotted grouper (Epinephelus coioides) (EcDDX3X). EcDDX3X encoded a 733-amino acid protein which shared 97% and 76% identity to spiny damselfish (Acanthochromis polyacanthus) and human (Homo sapiens), respectively. Amino acid alignment analysis showed that EcDDX3X contained conserved DExDc and Helic C domains. The transcription levels of EcDDX3X were significantly increased in poly I:C transfected cells and red-spotted grouper nervous necrosis virus (RGNNV) infected cells. Under fluorescence microscopy, the green fluorescence was observed evenly in the cytoplasm in EcDDX3X transfected cells. The ectopic expression of EcDDX3X significantly inhibited the replication of RGNNV, evidenced by the decreased numbers of the vacuoles evoked by RGNNV infection, and the reduced transcription levels of RGNNV coat protein (CP) and RNA-dependent RNA polymerase (RdRp) genes. In contrast, the replication of Singapore grouper iridovirus (SGIV) in grouper spleen (GS) cells was not significantly affected by EcDDX3X overexpression. Further studies showed that overexpression of EcDDX3X in vitro significantly increased the expression levels of several interferon associated cytokines or effectors. Moreover, the regulatory effect of EcDDX3X on interferon immune response was dependent on its N terminal region, but not the DExDc and Helic C domain. In addition, we also found that overexpression of EcDDX3X significantly increased the interferon promoter activity, and the activation of interferon immune response was regulated by both IRF3 and IRF7. Together, our results firstly showed that fish DDX3X exerted crucial roles in antiviral immunity against RNA virus infection via upregulating interferon antiviral responses.EcDDX3X was cloned and characterized.EcDDX3X encoded a cytoplasmic protein.EcDDX3X overexpression significantly inhibited the replication of RGNNV.EcDDX3Xoverexpression enhanced IRF3- and IRF7-mediated interferon response.