Abstract: Plant-specific WRKY transcription factors (TFs) are widely involved in various stress responses. However, their roles in abiotic stresses are still not well known in alfalfa (Medicago sativa). In this study, a WRKY gene, containing 1536 bp CDS length encoding a putative protein of amino acid 512, designated as MsWRKY33, was isolated from alfalfa. The alignment results revealed that the MsWRKY33 protein contains two conserved DNA-binding domains (WRKY domain) of 60 amino acids and a C2H2 zinc finger region (C-X4-C-X23-H-X-H), falling into group I of the WRKY protein. Protein localization prediction analysis indicated that MsWRKY33 is a nuclear-targeting protein. The expression of MsWRKY33 gene was up-regulated by salinity (NaCl), drought (PEG) and cold temperature (4℃), indicating that MsWRKY33 gene may be involved in the regulation of environmental stress responses in alfalfa. The fragment encoding 10-239AA was inserted into pET-30α (+) to construct the expression vectors, and SDS-PAGE analysis revealed that the MsWRKY33 protein could be expressed in prokaryotic cells. The full length cDNA of MsWRKY33 was amplified from alfalfa RNA and the plant expression vector pBI121-MsWRKY33 was constructed based on the pBI121 vector. Transgenic plants were obtained through somatic embryogenesis by Agrobacterium tumefaciens-mediated transformation. The npt Ⅱ gene and vector sequence were tested by PCR in the genome of transgenic plants and indicated that the target gene had been transferred. The qRT-PCR testing showed that the MsWRKY33 gene was over expressed in transgenic plants. This study will lay a foundation for further study of the function of the WRKY transcription factor in stress-tolerance regulation in alfalfa.