In this study, a novel NAC transcription factor was cloned and identified from Medicago sativa, designated as MsNAC3(GenBank No.：KC491186), Multiple alignment of amino acid sequences revealed that MsNAC3 has a high homology with MtNAC in Medicago truncatula and CarNAC5 in Cicer arietinum. which has a typical NAC conserved domain at the Nterminal as well as a highly variable Cterminal. A phylogenetic cluster analysis indicated MsNAC3 belonged to the ATAF subfamily of NAC proteins and having a close genetic relationship with MsNAC2(Medicago sativa L.) and BnNAC3 (Brassica campestris L.). A subcellular localization analysis in onion indicates that MsNAC3 is located in the nucleus. Expression analysis verified that MsNAC3 expression level has a significant increase under salt, drought, ABA and cold stresses. As well as, the expression of MsNAC3 in roots was higher than in leaves. The above results indicated that MsNAC3 may play an important role in the processes of stresssignal transduction as a positive regulatory factor.