Enterocytozoon bieneusi and Encephalitozoon spp. are microsporidian pathogens with zoonotic potential that pose significant public health concerns. To ascertain the occurrence and genotypes of E. bieneusi and Encephalitozoon spp., 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 of E. bieneusi, Encephalitozoon cuniculi, Encephalitozoon intestinalis and Encephalitozoon hellem, respectively. One red panda was detected positive for a mixed infection (E. bieneusi and E. 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 of E. bieneusi were found. Three genotypes of E. 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.