Simple SummaryMicrosporidia are obligatory intracellular fungi that cause infection in a broad variety of eukaryotes. However, there are few epidemiological studies on microsporidia in red pandas in China. Therefore, we conducted an epidemiological survey of microsporidia in red pandas from six zoos in Sichuan Province, China. In 198 fecal samples,Enterocytozoon bieneusiwas detected in 24 samples (12.1%), whileEncephalitozoonspp. was detected in 8 fecal samples (4.0%). Further analysis revealed the presence of genotypes ofE. bieneusiwith zoonotic concerns. Interestingly, we also observed that the infection rate of microsporidia was higher in semi-free red pandas with more frequent contact with other animals or humans. This is the first report on the occurrence and genotypes ofE. bieneusiandEncephalitozoonspp. in red pandas in Sichuan Province, China, contributing to our understanding of the worldwide distribution and genetic diversity of this pathogen.AbstractEnterocytozoon bieneusiandEncephalitozoonspp. are microsporidian pathogens with zoonotic potential that pose significant public health concerns. To ascertain the occurrence and genotypes ofE. bieneusiandEncephalitozoonspp., we used nested PCR to amplify the internal transcribed spacer (ITS) gene and DNA sequencing to analyze 198 fecal samples from red pandas from 6 zoos in China. The total rate of microsporidial infection was 15.7% (31/198), with 12.1% (24/198), 1.0% (2/198), 2.0% (4/198) and 1.0% (2/198) for infection rate ofE. bieneusi,Encephalitozoon cuniculi,Encephalitozoon intestinalisandEncephalitozoon hellem, respectively. One red panda was detected positive for a mixed infection (E. bieneusiandE. intestinalis). Red pandas living in semi-free conditions are more likely to be infected with microsporidia (χ2= 6.212, df = 1,p< 0.05). Three known (SC02, D, and PL2) and one novel (SCR1) genotypes ofE. bieneusiwere found. Three genotypes ofE. bieneusi(SC02, D, SCR1) were grouped into group 1 with public health importance, while genotype PL2 formed a separate clade associated with group 2. These findings suggest that red pandas may serve as a host reservoir for zoonotic microsporidia, potentially allowing transmission from red pandas to humans and other animals.