To investigate the structural characteristics and diversity of intestinal flora in rats with spleen deficiency diarrhea, twenty-four healthy rats were randomly divided into two groups, a control group and a model group. The model group was artificially replicated with a model of spleen deficiency diarrhea using the method of eating disorder and bitter cold. The highly variable V3-V4 region of the bacterial 16S rDNA gene of the rats intestinal contents was PCR-amplified using universal primers, and the amplicons were subjected to high-throughput sequencing using the Hiseq2500 platform. With bioinformatics analysis, the α diversity index indicates that there were not statistically significant differences in the intestinal microbial diversity between spleen-deficiency and diarrhea rats and normal rats. At the phylum level, the dominant phylums of the two groups were Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria. Compared with the control group, the relative abundance of Firmicutes, Actinobacteria, Proteo-bacteria and Spirochaetes in the model group increased, and the relative abundance of the Bacteroidetes was significantly reduced(P<0.05). At the genus level, the core flora of the two groups was Eubacterium_coprostanoligenes_group. Compared with the control group, the relative abundance of Eubacterium_coprostanoligenes_group and Jeotgalicoccus increased in the model group, and the increase of Jeotgalicoccus was significant(P<0.05); while the relative abundance of Romboutsia, Bacteroides and Odoribacter decreased, and Odoribacter decreased significantly(P<0.05). PICRUSt gene prediction showed that the intestinal microorganisms of rats were mainly metabolic functions, including sugar metabolism, amino acid metabolism, energy metabolism, cofactors and vitamin metabolism, but there were differences in various metabolic functions in the control group and model group. In conclusion, spleen deficiency diarrhea could cause changes of bacterial diversity in the intestine of rats, and significantly reduce the relative abundance of Bacteroidetes in the intestinal flora.