Enterocytozoon bieneusi is the most common species of microsporidia that infects humans and animals worldwide. However, no information is available on E. bieneusi infection among zoo animals in the Republic of Korea (ROK). Here, we investigated the prevalence of E. bieneusi among animals kept in zoos and the zoonotic potential of the E. bieneusi identified. E. bieneusi was detected only in one African lion (Panthera leo) with diarrhea, using PCR and sequencing analysis of the internal transcribed spacer (ITS) of the rRNA gene. A phylogenetic analysis based on the ITS gene showed that the lion isolate was classified into a novel genotype KPL belonging to Group 2. The KPL genotype identified in this study differed from genotype I in 6 nucleotides and from genotype I-like in 3 nucleotides, respectively, indicating that Group 2 has the capacity to infect a wide range of hosts. This is the first report of the presence of E. bieneusi in an African lion housed in a zoo in the ROK. Further investigation is necessary to study E. bieneusi infection among zoo animals in various regions and to determine the transmission route, in order to control E. bieneusi infection.