FTL (F-box Triple LRR ) protein belongs to the family of F-box proteins that play an important role in tolerance to low temperature. In this study, an MsFTL gene was isolated by candidate gene approach from alfalfa, and this gene was differently expressed in leaf under cold stress. The full-length cDNA of MsFTL gene was 1422 bp, which putatively encoded for 473 amino acids. By bioinformatic analysis, the MsFTL protein was found to carry an F-box domain and three LRR repeats in C-terminus. MsFTL showed homology closely to XP_003626345.1, a member of F-box/FBD/LRR-repeat protein in Medicago truncatula, with 11 differential sites between the two proteins. The expression of MsFTL was induced by low temperature, salt, drought stress and ABA treatments. We generated the transformation construct and transformed the MsFTL into tobacco. Under -4°C, wild-type tobacco leaves showed remarkable wilting, while the transgenic plants presented slightly stressed phenotype.Transgenic plants accumulated higher content of soluble protein and soluble sugar than that of wild-type. The activity of SOD and CAT in transgenic plants was higher than those of wild type under low temperature (4°C) for 24 h. Moreover, the MDA content in transgenic plants was lower than that of wild type. Thus, these results suggested that overexpressing MsFTL might significantly improve tobacco plant tolerance to low temperature stress.