Arsenic (As) pollution is a global problem, and the plant-based cleanup of contaminated soils, called phytoremediation, is therefore of great interest. Recently, transgenic approaches have been designed to develop As phytoremediation technologies. Here, we used a one-gene transgenic approach for As tolerance and accumulation in Arabidopsis thaliana . PvACR3, a key arsenite [As(III)] antiporter in the As hyperaccumulator fern Pteris vittata , was expressed in Arabidopsis , driven by the CaMV 35S promoter. In response to As treatment, PvACR3 transgenic plants showed greatly enhanced tolerance. PvACR3 transgenic seeds could even germinate and grow in the presence of 80 μM As(III) or 1200 μM arsenate [As(V)] treatments that were lethal to wild-type seeds. PvACR3 localizes to the plasma membrane in Arabidopsis and increases arsenite efflux into external medium in short-term experiments. Arsenic determination showed that PvACR3 substantially reduced As concentrations in roots and simultaneously increased shoot As under 150 μM As(V). When cultivated in As(V)-containing soil (10 ppm As), transgenic plants accumulated approximately 7.5-fold more As in above-ground tissues than wild-type plants. This study provides important insights into the behavior of PvACR3 and the physiology of As metabolism in plants. Our work also provides a simple and practical PvACR3 transgenic approach for engineering As-tolerant and -hyperaccumulating plants for phytoremediation.