Abstract. Streptococcus iniae is a causative agent of hemorrhagic septicemia in olive flounder, Paralichthys olivaceus, in Korea, resulting in serious economic losses. As a preventive measure, M VAC INIAE (Mastuken, Japan) was prepared from the S. iniae F2K strain and tested against the SI-36 strain prevalent on flounder fish farms on Jeju Island, Korea. F2K had a serotype of 38 (-) and SI-36 38 (+). The vaccine recognized both serotypes. It showed a very high effective immune response against S. iniae; the challenge test using the S. iniae SI-36 strain resulted in a relative percent survival (RPS) of 85.7-87.0% 2 weeks after vaccination and 71.0-80.0% 6 months after vaccination. Field vaccination and clinical challenge tests were performed at local Jeju aquafarms with S. iniae SI-36. These showed significantly reduced cumulative mortality when compared to the control group with RPS rates that ranged between 71-80%. Hence, the present study suggests that this vaccine showed a significant immune response against S. iniae and could be applied in commercial aquafarms as a therapeutic agent against â-hemolytic streptococcosis in cultured P. olivaceus.