Viral hemorrhagic septicemia (VHS) is one of the most serious viral diseases affecting farmed olive flounder (Paralichthys olivaceus) in Asian countries. VHS, caused by viral hemorrhagic septicemia virus (VHSV), occurs in over 80 different cultured and wild fish species worldwide. Our previous study demonstrated that VHSV infection can be restricted by adjusting the water temperature to over 17°C from the host optima. We confirmed that the effective VHSV immersion vaccine treatment was a tissue culture infection dose (TCID) of 105.5 TCID50/mL at 17°C. However, the safety of live VHSV immersion vaccines remains unclear. The objectives of this study were to 1) demonstrate the safety of the live VHSV immersion vaccine under co-habitant conditions and 2) estimate the pathogenicity of VHSV in live VHSV-vaccinated flounder at 10°C. No mortality was observed in olive flounder treated with the live VHSV immersion vaccine, and the vaccinated flounder challenged with VHSV did not transfer VHSV to naive fish at 10°C through cohabitation. VHSV titration was below the detection limit (< 1.3 log TCID50/mL) in live VHSV immersion vaccine-treated flounder challenged with VHSV at 10°C. This study demonstrated that flounder treated with the live VHSV immersion vaccine were resistant to VHSV inf...