Microbial biotransformations have a major impact on environments contaminated with toxic elements, including arsenic, resulting in an increasing interest in strategies responsible for how bacteria cope with arsenic. In the present work, we investigated the metabolism of this metalloid in the bacterium Ochrobactrum tritici SCII24. This heterotrophic organism contains two different ars operons and is able to oxidize arsenite to arsenate. The presence of arsenite oxidase genes in this organism was evaluated, and sequence analysis revealed structural genes for an As(III) oxidase (aoxAB), a c-type cytochrome (cytC), and molybdopterin biosynthesis (moeA). Two other genes coding for a two-component signal transduction pair (aoxRS) were also identified upstream from the previous gene cluster. The involvement of aox genes in As(III) oxidation was confirmed by functionally expressing them into O. tritici 5bvl1, a non-As(III) oxidizer. Experiments showed that the As(III) oxidation process in O. tritici requires not only the enzyme arsenite oxidase but also the cytochrome c encoded in the operon. The fundamental role of this cytochrome c, reduced in the presence of arsenite in strain SCII24 but not in an O. tritici DeltaaoxB mutant, is surprising, since to date this feature has not been found in other organisms. In this strain the presence of an aox system does not seem to confer an additional arsenite resistance capability; however, it might act as part of an As(III)-detoxifying strategy. Such mechanisms may have played a crucial role in the development of early stages of life on Earth and may one day be exploited as part of a potential bioremediation strategy in toxic environments.