ETHNOPHARMACOLOGICAL RELEVANCE: Xiangshao Sanjie Oral Liquid (XSSJ) is a traditional Chinese medicine formulation used clinically for the treatment of mammary gland hyperplasia (MGH), yet its mechanism remains obscure. AIM OF THE STUDY: The purpose of this study was to explore the therapeutic mechanism of XSSJ on MGH using a comprehensive strategy of plasma metabolomics and network pharmacology. MATERIALS AND METHODS: The rat model of MGH was established, and the multiple indicators were employed for efficacy evaluation. Then, a metabolomics strategy was established to find plasma metabolites and metabolic pathways that may be important in inducing MGH. In addition, UPLC-Q-TOF-MS and network pharmacological analysis were used to identify the prototype compounds of XSSJ in rat plasma and target genes that may cause the effect. Finally, the results were integrated and verified by RT-qPCR and Western Blot (WB). RESULTS: XSSJ has a therapeutic effect on MGH. 29 differential metabolites of XSSJ in the treatment of MGH were identified by metabolomics. After administration of XSSJ, 16 prototype compounds were found in the rat plasma, which were associated with 179 potential therapeutic targets. Comprehensive analysis revealed that XSSJ reversed the mRNA expression of EGFR, ESR1, AKT1, SRC and PTPN11 in MGH rats. In addition, different doses of XSSJ inhibited the expression of p-PI3K and p-AKT proteins. CONCLUSION: This study combined metabolomics and network pharmacology to reveal the regulatory effect of XSSJ on MGH through PI3K/AKT pathway, which provided further support for the clinical application of XSSJ.