Objective To establish a rat model for Blastocystis hominis infection and lay a foundation for the further study of B. hominis. Methods Total 30 male Sprague-Dawley rats were randomly divided into four infection groups and one control group with 6 in each group. In the infection groups, the rats were infected with 10⁵, 10⁶, 10⁷ and 10⁸ of B. hominis trophozoites, respectively, by oral gavage. In the control group rats were given the same volume of PBS only. Three days after infection fecal samples were collected until 18 days after infection with once every three days. Total 5 fecal samples were collected for each rat. The fecal samples were cultured in LES medium for 72 hours and B. hominis in the culture were observed under microscope and confirmed by PCR. The genotype of B. hominis before and after infection was identified by DNA sequencing of small subunit rDNA (SSU rDNA). The number of infected rats was calculated and clinical sign was observed. On the 21st day after infection, all the rats were euthanized and the contents of the intestines including duodenum, jejunum, ileum, colon and cecum were collected and cultured in LES for 72 hours before microscopic examination of B. hominis in the culture. The final infection rate and infection intensity were calculated. The collected intestinal tissues were sectioned and stained by Hematoxylin-Eosin(HE) to observer the infection and pathological changes. Pearson correlation was used for the statistic analysis. Results Total 2, 3, 5 and 6 rates were confirmed to be infected with B. hominis in the groups infected with 10⁵, 10⁶, 10⁷ and 10⁸ B. hominis trophozoites, respectively. All nfected rats had been identified to be infected with B. hominis on the 3rd day after infection except for the rats in group infected with 10⁵ trophozoites that in which the parasites were identified on the 6th day post infection. No rats got infected in the control group. Sequence analysis of SSU rDNA amplified from the infected parasites confirmed that the subtype of the infected B. hominis was ST7. There was no mutation within the amplified SSU rNDA for the parasite before and after infection in the rats. In vitro culture of the intestinal contents showed that B. hominis parasites were found in the ileum, cecum and colon. The number of B. hominis identified in cecum and the cultured parasite in cecum was correlated with those identified in the colons(r = 0.541, P < 0.05). No parasite was found in other parts of intestine. HE staining of intestinal sections showed that B. hominis and mucin were found in the section of cecum, however, there was no invasion of parasite into mucosa, or lesion or erosion or inflammatory cell infiltration in the mucosa identified. Conclusion A rat model was successfully established for infection of B. hominis in our lab by oral gavage with as less as 10⁵ trophozoites, however it is more stable to establish infection in rats using 10⁸ of trophozoites. The more the trophozoites used for the infection, the higher infection rate can be obtained.