Summary Environmental organoarsenicals are produced by microorganisms and are introduced anthropogenically as herbicides and antimicrobial growth promoters for poultry and swine. Nearly every prokaryote has an ars (arsenic resistance) operon, and some have an arsH gene encoding an atypical flavodoxin. The role of ArsH in arsenic resistance has been unclear. Here we demonstrate that ArsH is an organoarsenical oxidase that detoxifies trivalent methylated and aromatic arsenicals by oxidation to pentavalent species. Escherichia coli, which does not have an arsH gene, is very sensitive to the trivalent forms of the herbicide monosodium methylarsenate MSMA or MAs(V) and antimicrobial growth promoter roxarsone Rox(V), as well as to phenylarsenite PhAs(III), also called phenylarsine oxide or PAO. Pseudomonas putida has two chromosomally encoded arsH genes and is highly resistant to the trivalent forms of these organoarsenicals. A derivative of P. putida with both arsH genes deleted is sensitive to MAs(III), PhAs(III) or Rox(III). P. putida arsH expressed in E. coli conferred resistance to each trivalent organoarsenical. Cells expressing PpArsH oxidized the trivalent organoarsenicals. PpArsH was purified, and the enzyme in vitro similarly oxidized the trivalent organoarsenicals. These results suggest that ArsH catalyzes a novel biotransformation that confers resistance to environmental methylated and aromatic arsenicals. ArsH is an NADPH:FMN oxidoreductase that detoxifies the trivalent forms of the herbicide sodium methylarsenate (MSMA) and the poultry antimicrobial growth promoter roxarsone by oxidation to pentavalent species. ArsH is a tetrameric dimer of dimers with two FMN molecules bound in each dimer (graphic).