Raw fish consumption is increasing worldwide. Since around the year 2000, western regions of Japan have reported a foodborne disease of unknown cause that occurred after the consumption of flounder. In October 2010, a particularly large outbreak was reported in these regions among individuals who consumed flounder fish that had been raised in aquaculture systems. The median incubation period was 5 h (range, 4–19 h), and the most frequently reported symptom was diarrhea (80%). The risk estimate of the consumption of flounder was significantly higher than that of the development of symptoms (odds ratio = 9.50; 95% confidence interval, 1.59–∞). According to a trace-back investigation, all of the flounder responsible for the outbreak were raised in aquaculture systems. Microscopic examination revealed that the median amount of Kudoa septempunctata present in the muscle of flounder fish from the aquaculture farm was 4.5 × 10³ spores/g (range, 1.0 × 10³−9.6 × 10⁶ spores/g). The number of K. septempunctata spores required for the development of illness, as estimated using the Monte Carlo simulation, was 7.2 × 10⁷ spores/g; therefore, thus this might be the minimum ingestion threshold for the development of gastrointestinal symptoms. As a public health measure, the current study results should be referred to for the prevention of the gastrointestinal symptoms related to the consumption of flounder; the national public health authority has disseminated these results. We concluded that K. septempunctata-contaminated flounder fish were associated with the gastrointestinal symptoms of this recent outbreak.