Abstract BackgroundAmending from ancient classic, Ziyin Tongluo Formula (ZYTLF) has been prescribed to treat Postmenopausal osteoporosis (PMOP) for decades and obtained beneficial effect. However, the possible mechanisms of it are still unknown. MethodsOvariectomized rat model were established to validate the therapeutic effect of ZYTLF on PMOP by Micro-CT bone analysis and pathological observation. Subsequently, active ingredients of ZYTLF and corresponding putative targets were identified by online databases. Overlapping genes were obtained by mining genes associated with PMOP and then overlapping them with the putative targets. Key genes were selected from the multiple constructed and analyzed networks. GO and KEGG pathway enrichment analysis were performed by importing the key genes to the DAVID database. Moreover, validation of the binding association between key targets and their corresponding active compounds were accomplished by AutoDock Tools and other softwares. Lastly, Enzyme linked immunosorbent assay (Elisa) detection and Western blot analysis were utilized to further explore the possible mechanism of ZYTLF on PMOP. ResultsWith 129 target genes interacting with PMOP, 92 active compounds of ZYTLF corresponded to 243 targets, and 50 key genes were chosen. Network analysis revealed the top 10 active ingredients, such as quercetin, kaempferol and the top 50 key genes, such as ERα, p38 MAPK , p-AKT, TGF-β1. Enrichment analysis uncovered multiple signaling pathways, including estrogen signaling pathway, TNF signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway. Furthermore, our finding of the foremost active compounds were tightly bound to the core proteins was verified by molecular docking analysis. We confirmed that the prescription of ZYTLF could ameliorate the OVX-induced bone loss, suppress the osteoclast activity and boost osteoblastg ability through experimental studies. ConclusionThe potential mechanisms and therapeutic effects of ZYTLF against PMOP may be ascribed to inhabit osteoclast activity, boost osteoblast activity and enhance the expression of ERα .