BackgroundArsenic (As) with various chemical forms, including inorganic arsenic and organic arsenic, is the most prevalent water and environmental toxin. This metalloid occurs worldwide and many of its forms, especially arsenite [As(III)], cause various diseases including cancer. Organification of arsenite is an effective way for organisms to cope with arsenic toxicity. Microbial communities are vital contributors to the global arsenic biocycle and represent a promising way to reduce arsenite toxicity.MethodsBrevundimonassp. M20 with arsenite and roxarsone resistance was isolated from aquaculture sewage. ThearsHRNBCcluster and themetRFHHoperon of M20 were identified by sequencing. The gene encoding ArsR/methyltransferase fusion protein,arsRM, was amplified and expressed inEscherichia coliBL21 (DE3), and this strain showed resistance to arsenic in the present of 0.25–6 mM As(III), aresenate, or pentavalent roxarsone. The methylation activity and regulatory action of ArsRMwere analyzed using Discovery Studio 2.0, and its functions were confirmed by methyltransferase activity analysis and electrophoretic mobility shift assays.ResultsThe minimum inhibitory concentration of the roxarsone resistant strainBrevundimonassp. M20 to arsenite was 4.5 mM. A 3,011-bp arsenite resistancearsclusterarsHRNBCand a 5649-bp methionine biosynthesismetoperon were found on the 3.315-Mb chromosome. Functional prediction analyses suggested that ArsRMis a difunctional protein with transcriptional regulation and methyltransferase activities. Expression of ArsRMinE. coliincreased its arsenite resistance to 1.5 mM. The arsenite methylation activity of ArsRMand its ability to bind to its own gene promoter were confirmed. The As(III)-binding site (ABS) andS-adenosylmethionine-binding motif are responsible for the difunctional characteristic of ArsRM.ConclusionsWe conclude that ArsRMpromotes arsenite methylation and is able to bind to its own promoter region to regulate transcription. This difunctional characteristic directly connects methionine and arsenic metabolism. Our findings contribute important new knowledge about microbial arsenic resistance and detoxification. Future work should further explore how ArsRMregulates themetoperon and thearscluster.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-023-02876-z.