Sulfobacillus sp. TPY is a moderately thermophilic and acidophilic bacterium found in hydrothermal vents in the Pacific Ocean. This bacterium can oxidize ferrous sulfate (Fe(2+)) and elemental sulfur (S(0)) under separate conditions. We used random arbitrarily primed polymerase chain reaction (RAP-PCR) to screen and identify differentially expressed genes from bacteria grown on Fe(2+) or S(0) as the energy source. Fifty-five differential cDNA fragments were isolated and subjected to single-pass sequencing. Thirty-five fragments were identified as orthologs of known genes in the GenBank databases, of which 19 were confirmed to be differentially expressed at the transcriptional level by Northern blot analysis. Among these 19 genes, 14 genes, including isocitrate dehydrogenase, formyltetrahydrofolate deformylase, 3-hydroxybutyryl-CoA dehydrogenase, and GTP-binding protein, were upregulated in TPY grown on Fe(2+) or downregulated in TPY grown on S(0), while five genes such as the outer membrane adhesion-like protein, phosphomannomutase, and cysteine desulfurase sufS were upregulated in TPY strain grown on S(0) or downregulated in TPY grown on Fe(2+). These altered genes are involved in metabolism, osmotic stress, cell membrane alterations, oxidative stress, and the regulatory adaptive response. These results will aid our understanding of the molecular basis of Fe(2+) or S(0) oxidation by the moderately thermophilic and acidophilic bacteria.