ABSTRACT. Infection with Encephalitozoon cuniculi in rabbits frequently exists as a chronic, latent infection, and only a percentage of infected animals develop clinical disease. This study presents a seroepidemiological study of E. cunicucli infection in 337 pet rabbits collected from 20 prefectures in Japan in 2006 and 2007, using enzyme-linked immunosorbent assay (ELISA) capable of measuring IgG and IgM antibodies. These rabbits were divided into the following four groups: healthy and isolated rabbits (n=74, group I), healthy and companioned rabbits (n=121, group II), neurologically diseased rabbits (n=105, group III), and other diseased rabbits (n=37, group IV). Using ELISA for IgG antibodies, the highest detection rate, 81%, was seen in group III, the second highest, 75.2%, in group II, and the lowest, 29.7%, in group I, which was significantly different to the other groups except for group IV (43.2%). On the other hand, when ELISA was used for IgM antibody detection, 14-40% of rabbits in the four groups were also observed to have anti-E. cuniculi IgM. This study demonstrated high seroprevalence of E. cuniculi in not only neurologically diseased rabbits but also healthy and other diseased rabbits.