This is the first report of Enterocytozoon bieneusi in an equid species. Feces from 195 horses from 4 locations in Colombia were examined for E. bieneusi by polymerase chain reaction. Of these, 21 horses (10.8%) were found positive for E. bieneusi. The prevalence of E. bieneusi in horses <1 yr of age was significantly higher (23.7%) than in horses >1 yr of age (2.5%). No significant differences in prevalence were observed between male (13.7%) and female horses (9%). Sequencing of the internal transcribed spacer region of the SSUrRNA locus identified 3 genotypes. Two genotypes appear to be unique to horses and were named Horse 1 and Horse 2. A third genotype, identified as genotype D, was detected in 4 horses. This genotype, previously reported to infect humans, beaver, cattle, dogs, falcons, foxes, macaques, muskrats, pigs, and raccoons, is the most ubiquitous of the E. bieneusi zoonotic genotypes. Our findings indicate that E. bieneusi from horses can be a potential source of infection for humans.