NAC are specific novel transcription factors in plants with multiple biological functions, which play an important role in growth and development, anti-stress and hormonal regulation. In this study, the MsNAC1 gene in Medicago sativa L. was obtained by rapid amplification of cDNA ends (RACE) method. Bioinformatics analysis showed that MsNAC1 has an open reading frame (ORF) of 993 bp, encoding a stable, water-soluble protein with 330 amino acids. The MsNAC1 protein with a conserved NAM domain in the N-terminus and highly divergent C-terminus had the basic characteristics of the NAC transcription factors. It was predicted to locate in the nucleus with 2 nuclear localization sequences, and contain 9 glycosylation sites and 23 phosphorylation sites. The tertiary structure of MsNAC1 was predicted to be dimer. Sequence alignment analysis revealed that MsNAC1 has high homology with legume plants NAC protein but low with Gramineae. Phylogenetic analysis revealed that MsNAC1 was a member of NAC transcription factor family belonging to NAM subgroup, and has most close genetic relationship with GmNAC048. The expression of MsNAC1 under abiotic stresses was analyzed by qRT-PCR, its expression levels were upregulated in high-salinity, drought and cold stress treatments, indicating that this gene may be participate in regulation of plant abiotic stress response. The expression level in root was higher than that in leaf, and was highest and longest in root and leaf in cold stress treatment, indicating that this gene may be highly related to low temperature resistance in root.