Sulfur-oxidizing Acidithiobacillus thiooxidans has been shown to mediate ferric iron reduction at low pH under aerobic conditions. This was attributed to a nonenzymatic process involving sulfur intermediates formed during the sulfur oxidation and has already been successfully used for reductive bioleaching. However, the aerobic ferric iron reduction cannot be attributed to sulfur oxidation alone as hydrogen-grown At. thiooxidans also reduced ferric iron under aerobic conditions. Therefore, we compared the proteome profiles of ferric iron-reducing and nonreducing At. thiooxidans cells to reveal potential enzymes involved in the ferric iron reduction mechanism. The most elevated proteins in ferric iron-reducing cells were those involved in sulfur metabolism (sulfide quinone reductase, sulfur-reducing protein DsrE, oxidative cytochrome c-type SoxX), supporting the chemical mechanism of ferric iron reduction by inorganic sulfur compounds. In addition, redox proteins, including flavoproteins, FeS-containing proteins, and quinoproteins, which may play the role of iron reductases in the enzymatic mechanism of ferric iron reduction, were significantly increased. This study provides new insights into the mechanism of aerobic ferric iron reduction in sulfur-oxidizing At. thiooxidans, which involves an interplay of nonenzymatic and enzymatic processes. On the one hand, enzymatic processes promote the formation of sulfur intermediates, thereby reducing ferric iron nonenzymatically, which is further enhanced by enzymatic ferric iron reduction by redox proteins.