To investigate the differences in intestinal microbiota between diarrhea irritable bowel syndrome mice (IBS‐D) and healthy mice and to explore the effects of Jianpi mixture on intestinal microbes’ changes in IBS‐D mice based on 16S rDNA sequencing analysis. 48 young ICR male mice were randomly divided into four groups ( n = 12): (1) control group, (2) IBS‐D group fed with distilled water, (3) IBS‐D group fed with lactic acid bacteria compound, and (4) IBS‐D group fed with Jianpi mixture for 14 days. At the end of the treatment period, 5 mice were randomly selected from each group, and then the changes in intestinal microbiota in the mice before and after treatment were analyzed by 16S rDNA high‐throughput gene sequencing. Compared with the control group, the species richness and species diversity of intestinal microbiota in feces and intestinal mucosa of IBS‐D mice were decreased ( P < 0.05); IBS‐D mice showed changes in composition of and in ratio of the intestinal microbiota in feces and intestinal mucosa at the level of phylum, class, order, family, genus, and species. Treatment with Jianpi mixture increased the species diversity of intestinal microbiota in IBS‐D mice ( P < 0.05) and the abundance of beneficial bacteria ( P < 0.05) and decreased the abundance of harmful bacteria ( P < 0.05) at the level of phylum and genus. Compared with healthy mice, the species richness and species diversity of intestinal microbiota of IBS‐D mice are decreased. The intervention with Jianpi mixture can improve its diversity and regulate the equilibrium between beneficial and harmful bacteria.