Organoarsenical biotransformations are important components of the global cycling of arsenic. Roxarsone (3-nitro-4-hydroxybenzenearsenate or Rox(V)) and nitarsone (4-nitrobenzene arsenate or Nit(V)) are synthetic aromatic organoarsenicals used in the poultry industry as additives to prevent coccidiosis and improve feed efficiency. Here, we describe a novel pathway of resistance to roxarsone and nitarsone involving biotransformation of their trivalent forms (Rox(III)) and (Nit(III)) to the trivalent organoarsenicals HAPA(III) and pAsA(III), coupled to active extrusion of the aromatic aminobenezylarsenicals from the cells. The arsE, arsF, and arsG were cloned from the arsenic island in the chromosome of Shewanella putrefaciens 200. When expressed in Escherichia coli together, but not alone, arsEFG conferred resistance to Rox(III) and Nit(III) and decreased the accumulation of both. The cells transformed Rox(III) or Nit(III) to HAPA(III) or pAsA(III) by reducing the nitro group to an amine. Everted membrane vesicles from cells expressing arsG accumulated HAPA(III) or pAsA(III). Our data indicate that ArsE and ArsF together reduce Rox(III) or Nit(III) to HAPA(III) or pAsA(III), which are extruded from the cells by the efflux permease ArsG. Identification of the coupled pathway of ArsE, ArsF, and ArsG catalysis is a molecular description of a novel pathway for resistance to roxarsone and nitarsone.