Challenges of the Japanese flounder Paralichthys olivaceus with the monogenean Neoheterobothrium hirame were carried out to clarify the cause of the anemia, recently prevailing in the Japanese flounder in Japan. Hematological changes very similar to those previously reported in both wild and cultured anemic flounder were produced by the challenges with H. hirame. In the challenged flounder, the hemoglobin concentration decreased considerably and abnormal immature and mature erythrocytes having vacuolated or weakly stained cytoplasm, which characterize this anemia, appeared. The relationships among hemoglobin concentration, the ratio of immature erythrocytes and the above morphological changes in the blood smear of the challenged fish were very similar to those in the naturally affected anemic flounder. These hematological changes showed a clear correlation with the number of adult worms. These results, together with the previous findings, suggest that N. hirame is the main cause of the anemia prevailing in the Japanese flounder.