Abstract: Vitamin E is an essential nutrient for humans, but we cannot synthesize it by ourselves. Homogentisate phytyltransferase (HPT) is a key enzyme that determines vitamin E content. In this study, according to the sequence of MtHPT from Medicago truncatula, we cloned the Opening Reading Frame (ORF) of HPT from Medicago sativa by homology cloning strategy and termed MsHPT. NCBI blast results showed that it encoded a protein of 412 amino acid and belonged to the PT_UbiA superfamily. Multiple sequence alignment results showed that the similarity between the MsHPT protein and other HPT was 80%. In order to further study the gene, promoter sequences were cloned using genome walking technology. Analysis of the MsHPT promoter showed that defense and stress responsiveness cis elements, hormone (MeJA, ABA, GA and ethylene) responsiveness cis elements and light responsiveness elements presented in the promoter. Quantitative Real-time PCR results revealed that MsHPT was expressed throughout the plant, mainly in the leaves. Expression levels of MsHPT increased significantly in response to cold, NaCl, PEG, GA₃ and ABA treatments. The ORF of MsHPT was amplified with restriction enzyme recognition sites added, inserted into vector pBI121 and transformed into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation. According to the PCR results, seven positive lines were obtained. This study provides the basic information for further studies of the function of MsHPT in vitamin E biosynthesis and stress tolerance of M. sativa.