Arsenic (As) is a toxic carcinogen so it is crucial to decrease As accumulation in crops to reduce its risk to human health. Arsenite (As(III)) antiporterACR3 protein is critical for As metabolism in organisms, but it is lost in flowering plants. Here, a novel ACR3 gene from As hyperaccumulator Pteris vittata, PvACR3;1, was cloned and expressed in Saccharomyces cerevisiae (yeast), Arabidopsis thaliana (model plant), and Nicotiana tabacum (tobacco). Yeast experiments showed that PvACR3;1 functioned as an As(III)-antiporter to mediate As(III) efflux to an external medium. At 5 μM As(III), PvACR3;1 transgenic Arabidopsis accumulated 14–29% higher As in the roots and 55–61% lower As in the shoots compared to WT control, showing lower As translocation. Besides, transgenic tobacco under 5 μM As(III) or As(V) (arsenate) also showed similar results, indicating that expressing PvACR3;1 gene increased As retention in plant roots. Moreover, observation of PvACR3;1-green fluorescent protein fusions in transgenic Arabidopsis showed that PvACR3;1 protein localized to the vacuolar membrane, indicating that PvACR3;1 mediated As(III) sequestration into vacuoles, consistent with increased root As. Thus, our study provides a potential strategy to limit As accumulation in plant shoots, shedding light on engineering low-As crops to improve food safety.