Abstract: Late embryogenesis abundant (LEA) proteins are widely involved in many adverse situations for plants. In this study, we isolated a LEA4 gene from alfalfa by homology cloning strategy, termed MsLEA4-4. The MsLEA4-4 encoding revealed 512 amino acids and structure analysis showed that the gene contains eleven repeating TAQAAKEKTQQ amino acids sequences. Quantitative real-time PCR was used to detect MsLEA4-4 expression quantities in adverse situations. Results revealed that the expression of MsLEA4-4 was up-regulated by drought (PEG), salinity (NaCl), Cu²⁺, Zn²⁺ and ABA stress. Gene expression was highest at 2 h and 8 h under the stress of NaCl, Cu²⁺ and Zn²⁺. Under chilling and drought stress, the gene showed a rising trend. These results indicate that the MsLEA4-4 protein may be involved in the regulation of environment stress responses in alfalfa. The plant expression vector pCAMBIA3301-MsLEA4-4 was constructed and transgenic plants were obtained through infecting inflorescences of Arabidopsis by Agrobacterium tumefaciens-mediated transformation. The vector sequence was tested by antibiotic (PPT) and PCR in the genome of transgenic plants and indicated that the target gene had been transferred. This study provides basic information for further study of the MsLEA4-4 function in stress-tolerance regulation in alfalfa.