In previous investigations to treat acidic As(III)-containing solutions, an autotrophic acidophilic arsenite oxidizer, Acidiphilium acidophilum CJR1, has been isolated. Nonetheless, its physiological response toward As(III) remained unknown. An RNA-Seq approach was employed to obtain more insight. Though capable of autotrophic growth with arsenite, due to considerably better growth in the presence of yeast extract, A. acidophilum CJR1 was cultured under two heterotrophic conditions, one with additional arsenite and as a control one with yeast extract and 20 μM Fe2+. The results indicated the expression of arsenite oxidase genes under both conditions, suggesting that the enzyme may be constitutively expressed. In the As(III) condition, the COG categorization indicated that most strongly upregulated genes were energy-related or chaperones. These results provide the first transcriptomic insight into the arsenite response of an Acidiphilium acidophilum strain.