To determine the related metabolic pathways mediated by As(III) oxidation and whether AioR regulates other cellular responses to As(III), proteomic and genetic analyses were performed on the heterotrophic arsenite-oxidizing Agrobacterium tumefaciens GW4. The results showed that AioR was the main regulator for As(III) oxidation, and As(III) oxidation altered several cellular processes, especially phosphate, cell wall and carbon metabolism. In addition, the synchronous regulation of As resistance/oxidation proteins and phosphate transport proteins indicated that the metabolism of As was highly associated with that of phosphate. AioR suppressed the expression of the pst2/pho2 system and up-regulated the pst1/pho1 system to transport As and phosphate economically. AioR is the key driver of strain GW4 to adapt well in the As(III)-enriched sediment environment.