While the microbial community of the small intestine mucus (SIM) may also play a role in human health maintenance and disease genesis, it has not been extensively profiled and whether it changes with diet is still unclear. To investigate the flora composition of SIM and the effects of diet on it, we fed SD rats for 12 weeks with standard diet (STD), high-fat diet (HFD), high-sugar diet (HSD) and high-protein diet (HPD), respectively. After 12 weeks, the rats were sacrificed, SIM and stool samples were collected, and high-throughput 16S rRNA gene sequencing was used to analyze the microbiota. We found that fecal microbiota (FM) was dominated by Firmicutes and Bacteroidetes, while in SIM, Firmicutes and Proteobacteria were the two most abundant phyla and the level of Bacteroidetes dramatically decreased. The microbiota diversity of SIM was less than that of feces. The community composition of SIM varied greatly with different diets, while the composition of FM altered little with different diets. The relative abundance of Bacteroidetes and Allobaculum in SIM were negatively correlated with weight gain. There was no significant correlation between FM and weight gain. In conclusion, the community profile of SIM is different from that of feces and susceptible to diet.