Cryptosporidium and Giardia are ubiquitous protozoan parasites that infect a broad range of hosts. The presence of Cryptosporidium spp. and G. duodenalis was detected in 355 fecal samples of laboratory experimental rats from four experimental rat rearing facilities in China by PCR amplification of the small subunit (SSU) rRNA gene. The G. duodenalis positive samples were further characterized in the β-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes. The overall infection rates of Cryptosporidium spp. and G. duodenalis were 0.6% (2/355) and 9.3% (33/355), respectively, with no co-infection. Among the four facilities, only the rats in Zhengzhou1 were found positive for the two pathogens. Undetermined Cryptosporidium genotype was observed in one sample and C. ubiquitum in another sample. Assemblage G was identified in all the 33 G. duodenalis positive isolates at SSU rRNA gene, out of which 19, 20, and 21 isolates were also subtyped as assemblage G at tpi, gdh and bg gens, respectively. To our knowledge, this is the first report of Cryptosporidium and G. duodenalis infections in laboratory experimental rats in China. The infections of these pathogens in laboratory animals should be monitored routinely since they may interfere the biological experiments in these animals.