This study aims to explore the molecular mechanism of osthole (OST) on lipoteichoic acid (LTA) - induced mastitis in cows based on network pharmacology, molecular docking, and experimental verificatiom. Firstly, the set of OST targets was obtained by using databases such as PubChem, SwissTargetPrediction, UniProt, GeneCards, DrugBank, and DisGeNET, and the target genes related to BM were screened. Then, topological analysis was conducted through Venny, STRING database, and Cytoscape 3.8.0 software to establish the interaction network of drug target proteins and disease target proteins, and the core targets for treating BM were screened. Subsequently, GO functional and KEGG pathway enrichment analyses were performed using the Metascape database, and molecular docking and result visualization analyses of core proteins were carried out with AutoDock and Pymol. Finally, the effects of OST on the proliferation and inflammation of bovine mammary epithelial cells were detected by CCK-8 and fluorescence quantitative PCR experiments. The results indicated that there were 100 potential target proteins of OST, which intersected with 15,391 BM targets and yielded 88 key targets. Core targets such as Caspase-3 (CASP3), poly ADP-ribose polymerase 1 (PARP1), glycogen synthase kinase-3β (GSK3B), mitogen-activated protein kinase 14 (MAPK14), and recombinant histone deacetylase 2 (HDAC2) were screened out. KEGG pathway enrichment analysis revealed key pathways including hepatitis B, the pathogenesis of cancer, ligand-receptor interactions of neural activity, apoptosis, phosphatidylinositol 3 kinase-protein kinase B signaling pathway (PI3K-AKT), and cyclic adenosine monophosphate (cAMP) signaling pathway. Molecular docking results showed that the core targets had strong binding forces with OST, among which the binding activity with PARP1 was the strongest. In vitro results indicated that high, medium, and low concentrations of OST had inhibitory effects on inflammatory factors induced by LTA in bovine mammary epithelial cells, and the inhibitory effect was concentration-dependent, and it also inhibited the expression of PARP1 mRNA. In conclusion, this study initially revealed the mechanism of OST against BM, which was related to the network pharmacology and molecular docking predictions targeting PARP1. The expression of PARP1 mRNA in inflammatory bovine mammary epithelial cells was concentration-dependent on OST concentration. This provides a theoretical basis for the prevention and treatment of BM.