An outbreak of encephalitozoonosis occurred in a rabbit colony at a zoo in Japan. Throughout the two years after the onset, all 42 rabbits were investigated clinically, pathologically and serologically for prevention and control of the disease. Eleven rabbits (11/42, 26.2%) showed clinical symptoms. Of 38 rabbits examined to detect specific antibodies against Encephalitozoon cuniculi, 71.1% (n=27) were found seropositive; 20 out of 30 clinically healthy rabbits (except for 8 clinical cases) were seropositive. The infection rate was 76.2% (32/42), including 5 pathologically diagnosed cases. The results of serological survey revealed that asymptomatic infection was widespread, even among clinically healthy rabbits. However, encephalitozoonosis was not found by pathological examination in any other species of animals kept in the same area within the zoo. Isolation and elimination of the rabbits with suspected infection based on the results of serological examination were carried out immediately; however, encephalitozoonosis continued to occur sporadically. Therefore, all the remaining rabbits were finally slaughtered. Then, the facility was closed, and all the equipment was disinfected. After a two-month interval, founder rabbits were introduced from encephalitozoonosis-free rabbitries for new colony formation. Since then, encephalitozoonosis has not been seen in any animals at the zoo. In this study, biosecurity countermeasures including staff education, epidemiological surveillance and application of an "all-out and all-in" system for rabbit colony establishment based on serological examination were successfully accomplished with regard to animal hygiene and public health for the eradication of E. cuniculi.