Elemental sulfur (S(0)) oxidation in Acidithiobacillus spp. is an important process in metal sulfide bioleaching. However, the gene that encodes the sulfur dioxygenase (SDO) for S(0) oxidation has remained unclarified in Acidithiobacillus spp. By BLASTP with the eukaryotic mitochondrial sulfur dioxygenases (ETHE1s), the putative sdo genes (AFE_0269 and ACAL_0790) were recovered from the genomes of Acidithiobacillus ferrooxidans ATCC 23270 and Acidithiobacillus caldus MTH-04. The purified recombinant proteins of AFE_0269 and ACAL_0790 exhibited remarkable SDO activity at optimal mildly alkaline pH by using the GSH-dependent in vitro assay. Then, a sdo knockout mutant and a sdo overexpression strain of A. ferrooxidans ATCC 23270 were constructed and characterized. By overexpressing sdo in A. ferrooxidans ATCC 23270, a significantly increased transcriptional level of sdo (91-fold) and a 2.5-fold increase in SDO activity were observed when S(0) was used as sole energy source. The sdo knockout mutant of A. ferrooxidans displayed a slightly reduced growth capacity in S(0)-medium compared with the wild type but still maintained high S(0)-oxidizing activity, suggesting that there is at least one other S(0)-oxidizing enzyme besides SDO in A. ferrooxidans ATCC 23270 cells. In addition, no obvious changes in transcriptional levels of selected genes related to sulfur oxidation was observed in response to the sdo overexpression or knockout in A. ferrooxidans when cultivated in S(0)-medium. All the results might suggest that SDO is involved in sulfide detoxification rather than bioenergetic S(0) oxidation in chemolithotrophic bacteria.