【Objective】 bHLH transcription factors are numerous and can widely participate in plant growth, development, and stress processes. This experiment used Medicago truncatula R108 as the material to preliminarily explore the function of the MtbHLH25 gene. 【Methods】 Cloning of MtbHLH25 gene and promoter from Medicago truncatula using PCR amplification technology, construction of yeast expression vector, and transfer to Y2H Gold yeast strain using LiAc transformation method for yeast detection, construct subcellular localization vectors and transfer them into Agrobacterium EHA105 through freeze-thaw method. Inject the bacterial solution into tobacco epidermal cells and observe using SP8 laser confocal microscopy. The spatiotemporal expression level of MtbHLH25 gene in Medicago truncatula was studied using fluorescence quantitative PCR technology. 【Results】 The MtbHLH25 gene and promoter were successfully cloned from Medicago truncata, with a total length of 882 bp and encoding 293 amino acids. Promoter analysis revealed that it contains ABA response elements, MeJA response elements, GA response elements, and SA response elements. (2) The evolutionary tree showed that the MtbHLH25 protein is highly homologous with the bHLH protein in Vicia faba and Vicia villosa Roth. (3) The subcellular localization results showed that the MtbHLH25 protein was localized in the nucleus. (4) The yeast self activation test results showed that the MtbHLH25 protein has self activation activity. (5) The expression analysis results showed that MtbHLH25 was expressed in the roots, stems, leaves, flowers, and fruits of Medicago truncata, with the highest expression level in the roots; Exogenous SA, MeJA, ABA, GA, and salt stress all led to a decreasing trend in the expression of MtbHLH25 gene. It is speculated that SA, MeJA, ABA, GA, and salt stress have a negative regulatory effect on the expression of MtbHLH25 gene. Drought stress can significantly induce an increase in the expression level of MtbHLH25 gene, indicating that this transcription factor may play a positive regulatory role in drought stress. 【Conclusion】 The MtbHLH25 gene may be sensitive to salt stress and may play a positive regulatory role in drought stress. In addition, the MtbHLH25 protein has self activating activity and may have an activating effect on downstream promoter regulated reporter genes.