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Enterocytozoon bieneusi is an emerging zoonotic intestinal pathogen that infects humans and various animal species. Here, we aimed to determine the infection rate and genetic characteristics of E. bieneusi from bamboo rats from different regions of China using nested polymerase chain reaction-based amplification of the internal transcribed spacer region of the rRNA gene. A total of 435 bamboo rats fecal samples were collected from individual tank from Guangdong, Hunan, Jiangxi, Chongqing, and Guangxi, southeastern China. E. bieneusi was detected on 22 tanks (5.1%, 22/435), with a higher infection rate being observed among samples from Guangdong Province (10.9%, 5/46) compared with those from Hunan (9.3%, 10/107), Jiangxi (6.7%, 6/90), Chongqing (2.0%, 1/50), and Guangxi (0%, 0/142) (P <.01). Six genotypes were identified, including four known genotypes (D, EbpA, J, and PigEBITS7) and two novel genotypes (named BR1 and BR2). Of these, zoonotic genotype D was the most prevalent in the present study (n = 17). Phylogenetic analysis revealed that genotypes D, EbpA, and PigEBITS7 were clustered into Group 1, while genotypes J, BR1, and BR2 were clustered into Group 2. To our knowledge, this is the first report of E. bieneusi in bamboo rats. The identification of zoonotic genotype D as the predominant genotype in bamboo rats suggests that these animals represent a potential zoonotic risk for the transfer of the pathogen in China. • The bamboo rats represent a potential zoonotic risk for the transfer of E. bieneusi in China. • Four known genotypes and two novel genotypes were identified. • Z oonotic genotype D was the most prevalent in the present study. [ABSTRACT FROM AUTHOR]