[Objectives] Gut microbiota plays an important role in immune function and gastrointestinal system. The diversity and richness of gut microbiota are important physiological index to measure host health. Sex hormones play an important role on the growth and development of animals, but the effect of sex hormones on gut microbiota composition is not clear. [Methods] In this study, a mouse (Mus musculus) castration model was established by surgical method, fresh fecal samples of mice were continuously observed and collected during feeding, and the effects of sex hormones on the intestinal flora of mice were studied by 16S rRNA high-throughput sequencing technology. Usearch was applied to cluster reads with similarity above 97.0% and generate OTUs. Taxonomic annotations of feature sequences were processed by Bayesian classifier using SILVA as reference database. Statistics on composition in each sample were calculated at level of phylum, class, order, family, genus and species. QIIME was applied to obtain abundance of each species in samples, and distribution histogram at each taxonomic level were generated by certain R package. Alpha diversity metrics were evaluated by QIIME2. Beta diversity analysis was processed by QIIME. Blood was collected from the mice after they were fed for one month. The contents of estradiol and testosterone in serum of mice were determined by radioimmunoassay (RIA). [Results] Sex hormone levels in female and male mice decreased significantly after ovulation (Table 1). At phylum level, the intestinal bacterial communities of both normal mice and castrated mice were composed of Bacteroidetes, Firmicutes, Proteobacteria, Epsilonbacteraeota, Patescibacteria, Actinobacteria, Tenericutes, Deferribacteres, Acidobacteria and Cyanobacteria (Fig. 4). The populations of major bacteria found in normal and castrated mice were similar, with Firmicutes and Bacteroides as the dominant flora, and the sum of relative abundance percentages of the two flora was more than 80% (Table 4). Alpha diversity showed no significant difference in microflora evenness between the normal group and the castrated group (Table 5). Beta diversity showed no significant difference in microflora structure between the normal group and the castrated group. Followings are Beta diversity analysis based on four distance matrices: PCA analysis; PERMANOVA analysis and UPGMA analysis (Fig. 8). [Conclusion] The change of sex hormones did not cause the difference in the composition of the intestinal flora of mice.