Chloride (Cl-) ions cause major damage to crops in saline soils. Understanding the key factors that influence Cl- uptake and translocation will aid the breeding of more salt-tolerant crops. Here, using genome-wide association study and transcriptomic analysis, we identified a NITRATE TRANSPORTER 1 (NRT1)/PEPTIDE TRANSPORTER family (NPF) protein, GmNPF7.5, as the dominant gene locus influencing Cl- homeostasis in soybean (Glycine max). A natural SNP variation resulted in two haplotypes (GmNPF7.5HapA and GmNPF7.5HapB), which was associated with Cl- content. GmNPF7.5HapA mediated Cl- or nitrate (NO3-) uptake in a pH-dependent manner and exhibited higher permeability for Cl- over NO3-. The suppression of GmNPF7.5HapA expression decreased Cl- accumulation and salt damage in plants, whereas its overexpression showed the opposite effects. The elite haplotype GmNPF7.5HapB diminished Cl- transport activity independently from NO3- permeability, thus enhancing soybean salt tolerance. Furthermore, the protein kinase GmPI4Kγ4 could phosphorylate GmNPF7.5, which repressed Cl- uptake without affecting NO3- permeability. Our findings define a regulatory mechanism for Cl- control under NaCl stress, providing a strategy for the improvement of salt tolerance in soybean plants.