A newly developed nested polymerase chain reaction (PCR) assay targeting the internal transcribed spacer (ITS) region of the ribosomal DNA was applied to detect and characterize Encephalitozoon cuniculi DNA from pet rabbits in Japan. The analysis was carried out using 257 urinary samples and 314 fecal samples collected from 307 pet rabbits in the age group of 1 month to 12 years from 30 different prefectures of Japan and 107 fecal samples and 3 urinary samples collected from 1-month-old rabbits from 3 breeding facilities in Japan. We detected 840-bp amplicons in 20 urinary samples (7.78%) from the pet rabbits of the 13 prefectures and in 1 urinary (33.3%) and 6 fecal (5.6%) samples from the rabbits of the 2 breeding facilities. The sequences (803 bp) of the 27 amplicons had no variations and completely coincided with the sequence of E. cuniculi genotype I. To the best of our knowledge, this is the first report on the detection and genotype characterization of E. cuniculi DNA from pet rabbits in Japan.