This study aimed to investigate the mechanism of Ziziphi Spinosae Semen in inhibiting LPS-induced bovine mastitis using network pharmacology， molecular docking and in vitro experiments. The core components and potential targets of Ziziphi Spinosae Semen were screened through TCMSP， Swiss Target Prediction and Pharm Mapper databases， while bovine mastitis-related targets were obtained from DisGeNET， OMIM and GeneCards databases. Venny， STRING database and Cytoscape 3.8.0 software were used for topological analysis to construct compound-target and disease-target interaction networks. Hub targets for treating bovine mastitis were screened and subjected to GO functional and KEGG pathway enrichment analyses. Molecular docking and visualization were performed between hub targets and active components using AutoDock. An LPS-induced inflammatory model was established in bMECs， with CCK-8 assay， qRT-PCR， Western blot and ELISA used to analyze the effects of Ziziphi Spinosae Semen on inflammatory factors （IL-1β， IL-6， TNF-α） and hub targets at mRNA and protein levels. Results showed that： 9 bioactive components of Ziziphi Spinosae Semen and 235 potential therapeutic targets for bovine mastitis were identified； PPI analysis revealed 4 hub targets： epidermal growth factor receptor （EGFR）， estrogen receptor 1 （ESR1）， peroxisome proliferator-activated receptor gamma （PPARG）， and phosphatidylinositol-4，5-bisphosphate 3-kinase catalytic subunit alpha （PIK3CA）； GO and KEGG enrichment analyses indicated predominant involvement in biological processes including positive regulation of cytosolic ion concentration， receptor complex formation， and steroid binding， with significant associations to pathways such as central carbon metabolism in cancer， endocrine resistance， insulin resistance， and EGFR tyrosine kinase inhibitor resistance； Molecular docking demonstrated high binding affinity between ESR1 and the key components of Ziziphi Spinosae Semen， betulinic acid and daucosterol. In vitro experiments confirmed that Ziziphi Spinosae Semen bioactive components significantly suppressed the LPS-induced upregulation of IL-1β， IL-6， TNF-α， and ESR1、PI3KCA、PPARG mRNA and ESR1 protein levels in bMECs. In conclusion， Ziziphi Spinosae Semen exerts therapeutic effects against bovine mastitis primarily through bioactive components like betulinic acid by modulating key targets including ESR1. This study provides a scientific foundation for developing Ziziphi Spinosae Semen based therapeutic strategies for bovine mastitis intervention.