Enterocytozoon bieneusi is an emerging and clinically significant enteric pathogen in humans associated mainly with chronic diarrhea. It has been found in a variety of wild, domestic and companion mammals and birds. To date, epidemiological surveys of E. bieneusi infection in humans, other mammals and birds have been performed in more than 21 countries in Africa, the Americas, Australasia and Europe. In Asia E. bieneusi has been found in India, Thailand, Vietnam and Korea, but it has been quite unclear whether this pathogen is present in Japan. In the present study, we examined 149 DNAs extracted from 45 human (9 of them HIV-positive) and 104 animal fecal samples by PCR. Two dogs and a cat were positive and their genotypes were found to be dog specific and zoonotic (genotype K) types, respectively. Present study is the first record of E. bieneusi in Japan.