The full length cDNA coding for a novel vacuolar Na+/H+ antiporter (GmNHX1) was cloned from soybean and determined to consist of 2591 bp with a 5′-untranslated region of 464 bp, an open reading frame (ORF) of 1641 bp, and a 3′-untranslated region of 486 bp. The deduced protein sequence contains 546 aa with the typical characters of the vacuolar Na+/H+ antiporters, and shares high similarity with that of AtNHX1, OsNHX1 and AgNHX1. The soybean genome showed a single copy of the GmNHX1 gene. Northern blot analysis demonstrated that the expression of the GmNHX1 was tissue-specific, increased by ABA treatment, NaCl, KCl, LiCl and dehydration stress, and lower in leaves but higher in roots and hypocotyls of salt-tolerant than salt-sensitive cultivars. The GmNHX1 was overexpressed under the control of a tandem cauliflower mosaic virus (CaMV) 35S promoter in the model leguminous plant Lotus corniculatus L. and conferred salt-tolerance of the transgenic plants. Measurements of Na+ and K+ contents in both roots and shoots demonstrated that the plantlets of GmNHX1-overexpressing lines had lower Na+ and K+ content, and higher K+/Na+ ratio than the controlled lines that were transformed with the empty vector, which indicates that the salt-tolerance conferred by GmNHX1 is closely related with decreased accumulation of Na+ in the transgenic plants.