LGP2 is an important intracellular receptor that recognizes viral RNAs in innate immunity. To understand the mechanism of viral RNA recognition, we cloned an LGP2 cDNA and gene in Japanese flounder (Paralichthys olivaceus). Viral hemorrhagic septicemia virus-induced expressions of LGP2 mRNA were evaluated in vivo and in vitro by quantitative real-time PCR (Q-PCR) using primers based on the clone sequences. The expression of LGP2 mRNA in the kidney dramatically increased at 3 d postinfection. The expression of LGP2 mRNA also increased in the head kidney leukocytes stimulated with artificial dsRNA (polyinosin-polycytidylic acid) in vitro. To evaluate the antiviral activity of the flounder LGP2, three expression constructs containing pcDNA4-LGP2 (full-length), pcDNA4-LGP2ΔRD (regulatory domain deleted), and pcDNA4-Empty (as a negative control) were transfected into the hirame (flounder) natural embryo (hirame natural embryo) cell line. Forty-eight hours after transfection, the transfected cells were infected with ssRNA viruses, viral hemorrhagic septicemia virus, or hirame rhabdovirus. The cytopathic effects of the viruses were delayed by the overexpression of Japanese flounder LGP2. The Q-PCR demonstrated that mRNA expression levels of type I IFN and IFN-inducible genes (Mx and ISG15) in the hirame natural embryo cells overexpressing LGP2 were increased by polyinosin-polycytidylic acid and viral infections. These results suggest that Japanese flounder LGP2 plays an important role in the recognition of both viral ssRNA and dsRNA to induce the antiviral activity by the production of IFN-stimulated proteins.