[Objectives] This study systematically investigated the effects of pregnant mare serum gonadotropin (PMSG) on the gut microbiome in mice. [Methods] Twenty-four female mice were randomized into PMSG-treated (5 IU) and control groups (the same volume of sterile normal saline). Following 3-d intervention, we isolated the dominant strain Escherichia sp. GMJ-1 for in vitro growth curve modeling (Gompertz equation), performed full-length 16SrRNA sequencing (PacBio SMRT) of large intestine and small intestine contents, calculated α-diversity indexes (Shannon/Richness/Evenness) and β-diversity index (Unweighted UniFrac PCoA) via QIIME2 with species-level annotation, constructed bacterial networks (|r| > 0.6, P < 0.05) and core bacterial networks (|r| > 0.9, P < 0.01), and analyzed topology parameters. Statistical validation was conducted by t-test, one-way ANOVA (Tukey’s HSD), and PERMANOVA. [Results] The intestinal strain GMJ-1 showed significantly increased maximum growth rate under PMSG treatment (Fig. 1). In the large intestine, PMSG significantly increased the evenness of bacterial community (Fig. 2c) and drove a significant increase in alpha diversity (Fig. 2a). Even though PCoA demonstrated that intestinal segments exerted stronger influence on bacterial community structure than PMSG intervention (Fig. 3, PERMANOVA), PMSG induced the significant increase of Bacteroidetes and decrease of Proteobacteria at the phylum level (Fig. 4a) in the large intestine. Meanwhile, PMSG caused the enrichment of strains with potential metabolic benefits (Fig. 4b) in the large intestine, such as Eisenbergiella uncultured organism with the relative abundance increasing by 196 times. Network analysis revealed PMSG enhanced the complexity (nodes + 24%, edges + 41%) (Figs. 5a, b, Table 1) and the cohesion index (Fig. 6b) of the bacterial network in the large intestine. In addition, PMSG raised the proportion of positive correlation interactions among core bacterial species to 61.11%, increasing the positive relationship with Lactobacillus intestinalis and L. johnsonii as nodes (Figs. 7a, b). In the small intestine, the overall effects of PMSG on the diversity (Figs. 2a ~ c) and composition of the bacterial community at the phylum level (Fig. 4a) in the small intestine were not significant. At the species level, PMSG caused sharp fluctuations in some core bacterial species in the small intestine (Figs. 4c). For example, the relative abundance of Erysipelatoclostridium ramosum decreased by 90%. The robustness of the bacterial network in the small intestine was significantly reduced by PMSG (Fig. 6a), and the negative correlation interaction of the core bacterial species became dominant (53.85%) (Figs. 7c, d). [Conclusion] PMSG might drive the intestinal bacterial community to exhibit a segmental differentiated response of “being optimized in the large intestine - becoming fragile in the small intestine” through both direct promotion and indirect regulation mechanisms. This finding provides a theoretical basis for the development of scientific PMSG medication strategies tailored to animal intestinal segments.