Objective To explore the roles and effects of gonadotropin inhibitory hormone gonadotropin-inhibitory hormone (GnIH)/RF amide-related peptide-3 (RFRP-3) on the uterus through the hypothalamic-pituitary reproductive axis.    Methods  Ovariectomized estrogen primed (OEP) rats model was divided into GnIH injection group and normal saline injection group with 15 rats in each group, 2 g/L GnIH (16 μl/kg) and normal saline (16 μl/kg) were injected into the lateral ventricle of rats in the 2 groups respectively. 6 hours after injection, the uterine fluid of rats was obtained. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to separate the differential proteins in uterine fluid and UniProt was used to identify. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed, and the proteinprotein interaction (PPI) network of the differentially expressed proteins was constructed using Cytoscape 3.7.1 software. Hub proteins were detected from PPI network.    Results  The result  showed that the differentially expressed proteins separating by LC-MS/MS were 419 identified by UniProt. Among them, 279 were up-regulated and 138 were down-regulated. GO analysis showed that the differentially expressed proteins were mainly involved in response to organic substance, response to oxygen-containing compound, response to endogenous stimulus, response to stress. The top five enriched pathways obtained in the KEGG pathway analysis (P<0.05) were carbon metabolism, gap junction, long-term depression, regulation of actin cytoskeleton, biosynthesis of amino acids. Five hub proteins involved albumin（Alb），alpha-enolase 1(Eno1), peroxiredoxin-6 (Prdx6), tissue inhibitor of matrix metalloproteinases-1（Timp1）, Ras-related C3 botulinum toxin substrate 1 (Rac1) were obtained by analyzing PPI network.    Conclusion  The result  of this study indicate that GnIH may regulate the secretion of uterine cavity fluid protein through hypothalamic-pituitary reproductive axis. And regulate the physiological and pathological process of uterus by up-regulating Alb, Eno1, Prdx6 and down-regulating Timp1 and Rac1.