Sulfur-oxidizing bacteria (SOB) are the main microorganisms that participate in the natural sulfur cycle. To obtain SOBwith high sulfur-oxidizing ability under aerobic or anaerobic conditions, aerobic and anaerobic enrichmentswere carried out. Denaturing gradient gel electrophoresis (DGGE) profiles showed that the microbial community changed according to the thiosulfate utilizationduring enrichments, and Rhodopseudomonas and Halothiobacilluswere the predominant bacteria in anaerobic enrichment and aerobic enrichment, respectively,which mainly contributed to the thiosulfate oxidization in the enrichments. Based on the enriched cultures, six isolateswere isolated from the aerobic enrichment and four isolateswere obtained from the anaerobic enrichment. Phylogenetic analysis suggested the 16S rRNA gene of isolates belonged to the genus Acinetobacter, Rhodopseudomonas, Pseudomonas, Halothiobacillus,0chrobactrum, Paracoccus, Thiobacillus, and Alcaligenes, respectively. The tests suggested isolates related to Halothiobacillus and Rhodopseudomonas had the highest thiosulfate oxidizing ability under aerobic or anaerobic conditions, respectively; Paracoccus and Alcaligenes could aerobically and anaerobically oxidize thiosulfate. Based on the DGGE and thiosulfate oxidizing ability analysis, Rhodopseudomonas and Halothiobacilluswere found to be the main SOB in the sulfide-removing reactor, andwere responsible for the sulfur-oxidizing in the treatment system.