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 The B3 family genes constitute a pivotal group of transcription factors that assume diverse roles in the growth, development, and response to both biotic and abiotic stresses in plants. Medicago truncatula is a diploid plant with a relatively small genome, adopted as a model species for legumes genetics and functional genomic research. In this study, 173 B3 genes were identified in the M. truncatula genome, and classified into seven subgroups by phylogenetic analysis. Collinearity analysis revealed that 18 MtB3 gene pairs arose from segmented replication events. Analysis of expression patterns disclosed that 61 MtB3s exhibited a spectrum of expression profiles across various tissues and in the response to salt stress, indicating their potential involvement in salt stress signaling response. Among these genes, MtB3-53 exhibited tissue-specific differential expression and demonstrated a rapid response to salt stress induction. Overexpression of MtB3-53 gene in Arabidopsis improves salt stress tolerance by increasing plant biomass and chlorophyll content, while reducing leaf cell membrane damage. Moreover, salt treatment resulted in more up-regulation of AtABF1, AtABI3, AtHKT1, AtKIN1, AtNHX1, and AtRD29A in MtB3-53 transgenic Arabidopsis plants compared to the wild type, providing evidences that MtB3-53 enhances plant salt tolerance not only by modulating ion homeostasis but also by stimulating the production of antioxidants, which leads to the alleviation of cellular damage caused by salt stress. In conclusion, this study provides a fundamental basis for future investigations into the B3 gene family and its capacity to regulate plant responses to environmental stressors.