Abstract: This study discussed physiological regulation mechanism of salt resistance improvement of Medicago sativa L. (‘Zhongmu 3’ and ‘Longzhong’) by exogenous 2,4-epibrassinolide (EBR) in hydroponic conditions. The study analyzed the effects of exogenous EBR on the root growth, contents of osmotic adjustment substances, content of malondialdehyde (MDA), activities of antioxidant enzymes, contents of non-enzymatic antioxidant and active oxygen of M. sativa seedlings under NaCl stress. The result showed that under 150 mmol·L^-1 NaCl stress, root growth of ‘Zhongmu 3’ and ‘Longzhong’ seedlings were restrained significantly. Also root dry weight and root activity significantly decreased along with soluble protein content and antioxidant enzyme activities decreased in seedling root. On the other hand, active oxygen and MDA contents increased significantly along with the enhanced root lipid peroxidation while plasma membrane integrity was damaged. After addition of 0.1 μmol·L^-1 exogenous EBR, main root length, total root length, total root surface area, root volume, root tip number, root dry weight and root activity of the two alfalfa varieties significantly increased. Also soluble protein content, antioxidant enzymes (SOD, APX, GPX, CAT, GR) activities and non-enzymatic antioxidants (AsA, GSH) contents of seedling roots increased significantly. Then the production rate of superoxide anion free radicals, concentration of hydroxyl free radicals and contents of H₂O₂ and MDA decreased dramatically. Peroxidation of root lipid was relieved and the integrity of plasma membrane was enhanced. The above results illustrated that apart from mitigating the suppression effect of NaCl stress on alfalfa seedling root growth and enhancement of osmotic adjustment and antioxidant system activity, exogenous EBR promoted root growth and salt resistance of alfalfa. It also reduced active oxygen accumulation, membrane lipid peroxidation and oxidative damage on seedling root induced by NaCl stress.