For clarification of the etiological agent of the anemia recently prevailing in wild and cultured populations of the Japanese flounder Paralichthys olivaceus, flounder were challenged with the monogenean Neoheterobothrium hirame. In the challenged flounder, the hematological characteristics similar to those in the recent anemia were induced. Moreover, when N. hirame were artificially removed from the anemic flounder produced by the challenge, the hematological conditions recovered to normal levels. These results suggest that the parasite itself can induce anemia in Japanese flounder and refute a possibility that the parasite acts as a vector of other pathogens. They also indicate that the removal of N. hirame from anemic flounder is effective as a treatment of the anemia.