Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum leads to heavy mortality of ayu Plecoglossus altivelis in Japan. Previously, a 28°C warmed water treatment was shown to be an effective treatment for BCWD, since F. psychrophilum can not grow and maintain colonies at this temperature. However, it was unclear whether the bacteria might resuscitate after the treatment was over. Therefore, we investigated colony formation, membrane potential and pathogenicity of F. psychrophilum at various temperatures (15°C, 23°C, 28°C and 33°C) in sterilized underground water. Within 2 days at 28°C, F. psychrophilum completely lost their colony-forming abilities but still maintained their membrane potentials. It seemed that these cells entered into viable but non-culturable (VBNC) state. However, experimental ayu infection revealed that VBNC F. psychrophilum cells were unable to cause BCWD, suggesting that the cells were progressing towards death at 28°C.