Enterocytozoon bieneusi is closely linked with chronic diarrhea and wasting in AIDS. Although reported >10 years ago, little is known about the infection and the disease it induces in humans. Duodenal E. bieneusi spores from an AIDS patient were orally transmitted to 2 simian immunodeficiency virus-infected rhesus monkeys. Both animals began shedding spores within a week of inoculation, as confirmed by microscopy and polymerase chain reaction, and continued until euthanatized 7 and 8 months later. E. bieneusi infection in the gut was sparse, either because of moderate numbers of circulating CD4 cells or because monkeys are less susceptible than humans to this infection. This is apparently the first documented transmission of E. bieneusi infection between hosts.