Hirame rhabdovirus (HIRRV) was first reported from flounder (Paralichthys olivaceus) in Japan, and has recently been isolated from an aquaculture farm in the Tongyoung area of Korea. Total RNA isolated from RTG-2 cells infected with HIRRV was used in RT-PCR to obtain a cDNA coding for the glycoprotein of HIRRV Korean strain, CA-9703. Around half sized G protein in the C-terminal region was expressed as glutathione-S-transferase fusion protein, and used as viral vaccine against HIRRV. Flounder fry were vaccinated with low (50μg/mL) and high (300μg/mL) doses of vaccine by immersing them for 7 min in vaccine solution. After 1 month, the fish were challenged with HIRRV either by immersion or intraperitoneal injection. Specific losses of fish treated with low and high vaccine doses by intraperitoneal injection challenge were both 6.7%, in contrast to 43.3% in non-vaccinated fish. Losses of vaccinated fish by immersion challenge were 1.7% and 0% for low and high vaccine doses, respectively, and 15% for the non-vaccinated control.