As a typical type of abiotic stress, salt stress has become one of the core factors limiting plant growth and development. Diacylglycerol kinases (DGKs), as signaling enzymes, play a critical role in responding to abiotic stresses by phosphorylating diacylglycerol (DAG) to form phosphatidic acid (PA). In this study, the MsDGK5 gene was cloned from Medicago sativa L., and the overexpression vector pCAMBIA3301-MsDGK5 was constructed. We successfully obtained MsDGK5-overexpressing transgenic Arabidopsis plants using the floral dip method, and positive transgenic lines were selected for functional validation. Key findings include: (1) The full-length MsDGK5 gene spans 1428 bp, encoding 475 amino acids; (2) Phylogenetic analysis revealed that MsDGK5 is most closely related to MtDGK5 from Medicago truncatula; (3) The MsDGK5 gene is significantly induced by salt stress, and its expression level in roots reaches the highest at 48 hours after treatment; (4) Transgenic Arabidopsis overexpressing MsDGK5 exhibited enhanced salt tolerance, as evidenced by significantly higher fresh weight, survival rate, and total chlorophyll content, along with lower relative electrolyte leakage compared to wild-type (WT) plants. Studies have shown that overexpression of the MsDGK5 gene can significantly enhance the salt tolerance of Arabidopsis thaliana. The research results provide important theoretical basis for systematically analyzing the function of MsDGK5 gene in Medicago sativa and its molecular mechanism of salt stress response, and lay a molecular foundation for the genetic improvement and breeding of salt-tolerant alfalfa..