Soybeans, a crucial grain and oil crop, are valued for their high protein and oil content. Soil salinization is a significant abiotic stress that negatively impacts soybean growth and development, resulting in reduced yield and quality. The germination period represents a critical phase in soybean development. This study evaluated salt tolerance in 165 soybean mutant lines during germination, identifying five elite salt-tolerant germplasm resources. Multi-environment genome-wide association studies (GWASs) identified 11 significantly associated and 44 suggestive single nucleotide polymorphisms, alongside five novel quantitative trait loci linked to salt tolerance. Analysis of candidate regions qtl5-1 and qtl5-2 identified Glyma.05G097200 and Glyma.05G240200 as promising candidate genes, exhibiting distinct expression patterns between salt-tolerant and -sensitive genotypes. Functional characterization in Arabidopsis demonstrated that overexpression of the soybean gene GmMACPF1 induced salt sensitivity, while the macpf1 mutant of Arabidopsis displayed enhanced salt tolerance. Additionally, GmMACPF1 underwent selection during soybean domestication, with haplotypes Hap1 and Hap3 conferring improved salt tolerance. These results indicate that GmMACPF1 functions as a negative regulator of salt tolerance during germination, offering novel insights into the molecular mechanisms governing soybean response to salt stress during this crucial developmental stage.