The six- and seven-subunit Na+/H+ antiporters (Mrp) are widely distributed in bacteria. They are reported to be integral for pH homeostasis in alkaliphilic bacteria when adapting to high pH environments. In this study, operons encoding for the six-subunit Na+/H+ antiporters were found in the genomes of all studied Dietzia strains, which have different alkaline-resistant abilities. Disruption of the operon in the strain Dietzia sp. DQ12-45-1b which leads to declined growth in presence of hypersaline and alkaline conditions suggested that the six-subunit Na+/H+ antiporter played an important role in hypersaline and alkaline resistance. Although the complexes DqMrp from DQ12-45-1b (strain with high alkaline resistance) and DaMrp from D. alimentaria 72(T) (strain with low alkaline resistance) displayed Na+(Li+)/H+ antiport activities, they functioned optimally at different pH levels (9.0 for DQ12-45-1b and 8.0 for 72(T)). While both antiporters functioned properly to protect Escherichia coli cells from salt shock, only the DqMrp-containing strain survived the high alkaline shock. Furthermore, real-time PCR results showed that the expression of mrpA and mrpD induced only immediately after DQ12-45-1b cells were subjected to the alkaline shock. These results suggested that the expression of DqMrp might be induced by a pH gradient across the cell membrane, and DqMrp mainly functioned at an early stage to respond to the alkaline shock.