Multidrug and toxic compound extrusion transporters (MATE) are able to transport a wide variety of substrates such as metals, hormones and secondary metabolites, and they thus play an important role in growth and development in plant. In this study, the full-length coding sequence of MsMATE1 (NCBI GeneBank accessions: MN547958) and its promoter pMsMATE1 (MT505313) were isolated based on genome and transcriptome sequences of Medicago sativa under iron deficiency. The MsMATE1 is 1470 bp which encodes a putative protein of 489 amino acids. According to bioinformatics analysis, the MsMATE1 protein contains a typical MATE_like superfamily domain, which belongs to the eukaryotic subtype; MsMATE1 is a transmembrane protein, and the main component of its secondary structure is α helix. Phylogenetic analysis showed that MsMATE1 protein is closely related to MtMATE1 (XP013453190.1). The pMsMATE1 is 1598bp which were detected with multiple elements responding to hormone and abiotic stress. MsMATE1 was expressed in roots, shoots and leaves of alfalfa seedlings with the highest abundance in shoots. Under iron-deficient or iron-excess stresses, MsMATE1 was significantly up-regulated in different tissues of alfalfa seedlings, with the highest up-regulation in shoots. In MsMATE1 transgenic tobacco plants if compared with those of wild type plants, the activities of SOD, POD, and CAT, chlorophyll content and soluble protein content were significantly increased, while the significant reduction on content of MDA was observed under iron-deficient or iron-excess conditions, compared with those of wild type plants.The results showed that heterologous expression of MsMATE1 improved the tolerant against iron-deficient or iron-excess stresses in plants. MsMATE1 might be a candidate gene for improving tolerance to iron stress by genetic engineering. Collectively, this study laid a foundation for future deciphering the MsMATE1-involved molecular mechanism upon iron stresses.