<p>Arsenic is a toxic metalloid. It is released to the environment normally as arsenite [As(III)], largely due to mining activities. As(III) is more toxic and in many acidic mining environments more mobile than arsenate [As(V)] and therefore the oxidation of As(III) to As(V) is fundamental to immobilize and treat arsenic waste. Here, we report on the isolation and the phenotypic as well as genomic characterization of a novel extremely acidophilic As(III)-oxidizing strain from Reiche Zeche, a former silver mining site (Freiberg, Germany). The isolated strain CJR1 was identified as <i>Acidiphilium acidophilum</i> by 99.7% 16S rRNA positions shared with the type strain <i>A. acidophilum</i> DSM 700^T and by the Type (Strain) Genome Server platform. The new isolate is able to grow lithoautotrophically with As(III) from pH 4.9 to 1.5 with an optimum around pH 2.0. However, growth and As(III) oxidation rates were improved by adding yeast extract. The genome of CJR1, with 5.17 Mbp, is considerably bigger than that of other <i>Acidiphilium</i> strains and comprises four circular plasmids. The genome suggests the presence of the Calvin–Benson–Bassham cycle of multiple arsenic resistance operons (<i>ars</i>) and of an arsenite oxidase (<i>aio</i>).</p> Graphical Abstract <p>The representation of the genome seen on the graphical abstract was made using GenoVi [<CitationRef CitationID="CR1">1</CitationRef>].</p>