Background: The occurrence of serious diseases, such as inflammatory diseases and cancer, in the small intestine is significantly lower than that in the colon. The differentiation of small-intestine microbiota from large-intestine microbiota might hold great significance. Materials and methods: To compare floral composition and functions between the two types of microbiota, ileal contents and feces were collected from Sprague Dawley (SD) rats, and the V3–V4 region of the 16S ribosomal ribonucleic acid (rRNA) gene in these rats was amplified and sequenced. We subjected the data to bioinformatics analyzing. Results: Compared with feces, about 50% of bacterial genera in the ileum were exclusive, with low abundance (operational taxonomic units [OTUs] P < 0.05). A high-fat diet (HFD) and specific-pathogen–free (SPF) conditions had a profound influence on α-diversity and abundance but not on the exclusive/shared features or β-diversity of samples. Intestinal microbiota were composed of high-prevalence dominant, low-prevalence dominant, and less-dominant bacteria. Conclusions: The present findings suggested that ileal and fecal bacteria were different structurally and functionally. These differences might be key to the fundamental protection of the small intestine from diseases.