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In this study, genomic DNA was screened from Halobacillus andaensis NEAU-ST10-40T by selection in Escherichia coli KNabc lacking three major Na+/H+ antiporters. One gene designated upf0118 exhibiting Na+(Li+)/H+ antiport activity was finally cloned. Protein alignment showed that UPF0118 shares the highest identity of 81.5% with an unannotated gene encoding a protein with uncharacterized protein function belonging to UPF0118 family from H. kuroshimensis, but shares no identity with all known specific Na+(Li+)/H+ antiporter genes or genes with Na+(Li+)/H+ antiport activity. Growth test, western blot and Na+(Li+)/H+ antiport assay revealed that UPF0118 as a transmembrane protein exhibits pH-dependent Na+(Li+)/H+ antiport activity. Phylogenetic analysis indicated that UPF0118 clustered with all its homologs belonging to UPF0118 family at a wide range of 22-82% identities with the bootstrap value of 92%, which was significantly distant with all known specific single-gene Na+(Li+)/H+ antiporters and single-gene proteins with the Na+(Li+)/H+ antiport activity. Taken together, we propose that UPF0118 should represent a novel class of Na+(Li+)/H+ antiporter. To the best of our knowledge, this is the first report on the functional analysis of a protein with uncharacterized protein function as a representative of UPF0118 family containing the domain of unknown function, DUF20. [ABSTRACT FROM AUTHOR]