Increasing levels of sodium carbonate are major causes of grassland alkalinization in the Song- Nen Plain of northeast China. In winter-spring alternation, plants are often subjected to double stresses of alkaline salt and freeze-thaw. Here, Medicago sativa CV. Zhongmu No.1 seedlings were used to study the effects of laboratory-simulated basic salt (BS) and freeze-thaw (FT) stresses on the contents of osmoregulatory substances, biological membrane permeability, and antioxidant enzyme activity. The results showed that under individual BS- or FT- stress, the soluble protein in seedlings decreased by 9.6-20.2%, proline and MDA contents increased by 18.4-48.1 and 7.8-37.7%, respectively. Furthermore, the activities of SOD and POD increased by 6.2-24.9 and 4.8-30.3%, respectively. During the freezing and thawing cycles, both proline and MDA contents initially increased and then decreased; these observations contrasted with those for soluble protein content. Activities of SOD and POD increased in attempts to resist reactive oxygen species (ROS). Moreover, M. sativa CV. seedlings under combined stress of BS and FT showed significantly higher physiological responses than those under individual stress (BS- or FT- stress). The results of this study demonstrate the molecular basis for BS- and FT- stress tolerance by M. sativa. In turn, this could lead to strategies for further enhancement of these tolerances in the plant.