The purpose of this study is to investigate the regulation of the immune function of canine bone marrow mesenchymal stem cells (BMSCs) by berberine hydrochloride (Ber) through the PD-1/PD-L1 signaling pathway. Method: Network pharmacology was used to screen the target of berberine in BMSCs, and GO function and KEGG pathway enrichment analysis were performed. Using dog BMSCs as research materials, berberine was added to the culture medium, and cell viability and aging were evaluated using CCK-8 and SA - β - gal staining. ELISA was used to detect sPD-L1, sPD-L2, IL-10, IL-6, IL-4, TNF - α secretion levels, and RT qPCR was used to detect PD-L1, STAT3, AKT1, JAK2, MTOR, TLR4, RELA, MAPK14 expression levels. As a result of network pharmacology screening, 82 common targets of berberine were identified in dog BMSCs. KEGG pathway enrichment analysis showed that 9 key targets of berberine, including PD-L1, PD-L2, STAT3, AKT1, JAK2, MTOR, TLR4, RELA, and MAPK14, were enriched in the PD-1/PD-L1 immune pathway; CCK-8 and SA - β - gal analysis showed that treatment with 0-20 μ M berberine for 48 hours did not significantly reduce the proliferation activity of BMSCs in dogs; The ELISA results showed that berberine at a concentration of 10-20 μ M for 48 hours promoted the synthesis and secretion of sPD-L1 and sPD-L2, while inhibiting the synthesis and secretion of IL-6 and TNF - α, with no significant effect on IL-4 and IL-10. The RT qPCR results showed that after treatment with berberine, the expression level of PD-L1 gene was upregulated, TLR4, RELA, and MAPK14 were downregulated, while the expression levels of STAT3, AKT1, JAK2, and MTOR remained unchanged. Conclusion: Berberine enhances the immune regulatory function of canine BMSCs through the PD-1/PD-L1 pathway, providing a theoretical basis for the clinical application of canine BMSCs and their combination with berberine.