Directly relevant to the toxicity, mobility, and fate of arsenic, the biotransformation of inorganic and organic arsenicals has been extensively concerned, including roxarsone, a widely applied organoarsenical feed additive in poultry industry. Yet, little is known about the transformation details of roxarsone in microbial fuel cells (MFC). In this study, a two-chambered Shewanella oneidensis MR-1 microbial fuel cell was employed to investigate the transformation processes of roxarsone at various carbon source levels. Results show that limited carbon source remarkably inhibited inorganic arsenic release along roxarsone transformation, whereas numerous arsenical species were detected to be released into systems with sufficient carbon source supply, including trivalent and pentavalent inorganic arsenics, monomethylarsonous acid (MMA), and 4-hydroxy-3-aminobenzene arsonic acid (HAPA). Shewanella oneidensis MR-1 was able to cleave the C-As bond of trivalent HAPA yielding inorganic arsenics and MMA, even in the absence of the arsI gene encoding ArsI C-As lyase. We proposed a two-step nitro- and pentavalent-arsenate group reduction pathway for the roxarsone bioelectrochemical transformation. In addition, results indicated that the attached cells onto the electrode surface played a key function in the two-step reduction of roxarsone to trivalent HAPA, whereas planktonic cells were most likely responsible for the C-As bond breakage and the following dearylation. With these qualitative and quantitative estimations, it provides new insights into the mechanistic understanding of the roxarsone biotransformation process in microbial fuel cells, which is important for the biogeochemical cycling of arsenic.