In this study, the MfMYB30 gene was cloned from Medicago falcata L. cv. Hulunbuir by RTPCR based on transcriptomic sequence, and the gene expression was analyzed by bioinformatics. The results showed that: (1) the ORF sequence length of MfMYB30 gene was 957 bp, encoding 318 amino acids, with molecular weight of 86.85 KD and theoretical isoelectric point of 5.11. MfMYB30 protein is a hydrophobic protein without transmembrane structure and signal peptide sequence. (2) Phylogenetic analysis showed that the MfMYB30 protein of M.falcata clustered into a group with close genetic relationship with MsMYB4 of Medicago sativa, AtMYB30 of Arabidopsis thaliana, CcMYB30 of Caja caja, MtMYB30 of Medicago truncatula and GmMYB60 of Glycine max. (3) Realtime quantitative PCR results showed that the relative expression level of MfMYB30 decreased first and then increased after different days of simulated cutting, and reached the peak value after 7 days of cutting. (4) By analyzing the subcellular localization of protoplasts in A. thaliana, it was found that the protein was located in the nucleus. It is speculated that MfMYB30 gene may play an important role in the response to cutting or grazing stress of M.falcata.