Alfalfa (Medicago sativa L.) is an important and widely cultivated forage grass. The productivity and forage quality of alfalfa are severely affected by salt stress. Melatonin is a bioactive molecule with versatile physiological functions and plays important roles in response to various biotic and abiotic stresses. Melatonin has been proven efficient in improving alfalfa drought and waterlogging tolerance in recent studies. In our reports, we applied melatonin exogenously to explore the effects of melatonin on alfalfa growth and salt resistance. The results demonstrated that melatonin application promoted alfalfa seed germination and seedling growth, and reduced oxidative damage under salt stress. Further application research found that melatonin alleviated salt injury in alfalfa plants under salt stress. The electrolyte leakage, malondialdehyde (MDA) content and H 2 O 2 content were significantly reduced, and the activities of catalase (CAT), peroxidase (POD), and Cu/Zn superoxide dismutase (Cu/Zn-SOD) were increased with melatonin pretreatment compared to control plants under salt stress with the upregulation of genes related to melatonin and antioxidant enzymes biosynthesis. Melatonin was also involved in reducing Na + accumulation in alfalfa plants. Our study indicates that melatonin plays a primary role as an antioxidant in scavenging H 2 O 2 and enhancing activities of antioxidant enzymes to improve the salt tolerance of alfalfa plants.Plants 2020, 9, 220 2 of 17 world's irrigated land affected by saline or sodic globally [7,8]. Salinity has become one of the most important environmental stress factors impairing worldwide agricultural productivity. Under salinity conditions, the seed germination, growth and development processes of alfalfa are inhibited and they finally impair the herbage yield, as well as the forage quality [9]. Breeding new alfalfa cultivars with high salt tolerance is always needed. Genetic engineering and conventional breeding have been proven efficient for improving salt tolerance of various plant species [10][11][12][13]. However, they are time-consuming and complicated. Exogenous application of certain plant growth regulators such as phytohormone and other small molecules has been proven efficient at overcoming the harmful effects of salt stress on plants [14][15][16][17]. Moreover, plant growth regulators in low concentrations always play a role in plants, and are cost-effective. Foliar spraying of salicylic acid (SA) on faba bean inhibits Na + accumulation and lipid peroxidation, improving the antioxidant resistance and finally mitigating the damage caused by salinity [18]. Exogenously applied poly-γ-glutamic acid on wheat maintains the Na + and K + homeostasis and enhances antioxidant capacity by alleviating salinity damage under salt stress conditions [19]. Exogenous spermidine application to salt-stressed cucumber improves the photosynthetic capacity and the activity of key enzymes for CO 2 fixation by regulating the expression of related genes, and tolerance to salinity is thus...