Abstract: The aim of this study was to explore the salt resistance and physiological regulation mechanisms induced by exogenous 2,4-epibrassinolide (EBR) in alfalfa (Medicago sativa) seedlings. The effect of EBR on photosynthetic pigment contents, gas exchange parameters, and ion uptake, transport, and disputation in seedlings of M. sativa cv. Zhongmu No.3 and M. sativa cv. Longzhong under NaCl stress were investigated using a hydroponic method. In the 150 mmol/L NaCl stress treatment, alfalfa seedling leaves showed significant decreases in photosynthetic pigment contents and photosynthetic ability, and disrupted metabolic processes of ion uptake, transport, and disputation. When EBR at 0.1 μmol/L was supplied to seedlings under NaCl stress, the chlorophyll a, b, and a+b contents increased, the chlorophyll a/b increased, and the chlorophyll/carotenoid decreased. Also, the photosynthetic rate, transpiration rate, stomatal conductance, water use efficiency, and photosynthetic ability increased after 0.1 μmol/L EBR treatment. These changes would facilitate the accumulation of aboveground and underground biomass. In the NaCl-stressed seedlings treated with 0.1 μmol/L EBR, the Na⁺ and Cl^- contents and the Cl^-/Na⁺ in different organs (leaf, stem, and root) significantly decreased, and the contents of K⁺, Ca²⁺, and Mg²⁺ and the K⁺/Na⁺, Ca²⁺/Na⁺, and Mg²⁺/Na⁺ significantly increased. The transport ratio of inorganic ions and transport selectivity ratio of cations were effectively regulated in the seedlings treated with EBR. Together, these results show that EBR treatment effectively alleviated photo inhibition caused by NaCl stress in alfalfa seedlings, and promoted the synthesis and accumulation of organic matter. Treatment with EBR also improved the selective absorption and transport of inorganic ions, helping to maintain the metabolic ion balance and improve the NaCl-resistance of alfalfa seedlings.