BACKGROUND: Mesenchymal stem cells (MSCs) possess a variety of immunomodulatory functions that can vary depending on the MSC line. Investigating priming strategies is essential for increasing the immunomodulatory potential of MSCs. METHODS: Human clonal MSCs (cMSCs) were primed with TNF-α, IFN-γ, IL-1β, IFN-α, and vitamin B6. Their immunomodulatory functions, including T-cell proliferation and cytokine production, were analyzed. The primed cMSCs were injected intravenously into a mouse model of ovalbumin-induced atopic dermatitis (AD), and their therapeutic effects were evaluated. RESULTS: We identified IFN-α and vitamin B6 as promising priming agents when they are combined with TNF-α and IFN-γ. The primed cMSCs showed expression of galectin-9 (Gal-9), IL-1Ra, and PDL-1. Gal-9 facilitates the induction of regulatory T cells (Tregs) and apoptosis. Treatment with primed cMSCs significantly alleviated pathological changes in an AD mouse model. Notable improvements included a reduction in epidermal thickness (p  < 0.05), a decreased number of mast cells and eosinophils in the dermis (p  < 0.01), restored expression of claudin-1 in the epidermis (p  < 0.0001), and lower serum levels of IgE (p  < 0.05). CONCLUSIONS: This novel combination of priming factors significantly promotes the immunomodulatory functions of cMSCs by inducing Gal-9. Consequently, Gal-9 may serve as an excellent biomarker for screening primed cMSCs for their immunomodulatory capabilities, facilitating a more accurate assessment of their therapeutic effectiveness.