The Arabidopsis vacuolar Na+/H+ antiporter gene, AtNHX1, was introduced into soybean by Agrobacterium-mediated transformation. Four independent kanamycin resistant lines were obtained. The result of PCR, Southern blotting and Northern blotting analyses demonstrated that the AtNHX1 gene was successfully inserted into the soybean genome and stably expressed in these kanamycin resistant lines. The stability of AtNHX1 expression and salt resistance were evaluated in the soybean transformants for over 6 generations. Two independently derived transgenic lines with high expression level of AtNHX1 were selected, and propagated to generation T-5 in the absence of selection pressure. PCR and RT-PCR examinations revealed that AtNHX1 was highly expressed in all investigated transgenic T-5 progenies. Furthermore, all transgenic T-5 plants showed resistant to salt stress, same as those of homozygous T-2 plants. Taken together, our results indicated that constitutive expression of AtNHX1 enhanced salt tolerance in soybean for over 6 generations, suggesting a great potential use of AtNHX1 for improving salt tolerance in plants by genetic engineering.