Chromate (Cr­VI) is a highly phytotoxic contaminant that is ubiquitous in soils. However, how Cr­(VI) is taken up by plant roots remains largely unknown. Here, we show that the high-affinity sulfate transporter Sultr1;2 is responsible for Cr­(VI) uptake by the roots of Arabidopsis thaliana. Sultr1;2 showed a much higher transport activity for Cr­(VI) than Sultr1;1 when expressed in yeast cells. Knockdown of Sultr1;2 expression in Arabidopsis markedly reduced the Cr­(VI) uptake rate, whereas knockout of Sultr1;1 had no or little effect. A double-knockout mutant (DKO) of the two genes lost the ability of Cr­(VI) uptake almost completely. The Sultr1;2 knockdown mutant or DKO plants displayed higher resistance to Cr­(VI) under normal sulfate conditions as a consequence of the lower tissue Cr accumulation. Overexpression of Sultr1;2 substantially increased Cr­(VI) uptake with shoot Cr concentration being 1.6–2.0 times higher than that in the wild-type. These results indicate that Sultr1;2 is a major transporter responsible for Cr­(VI) uptake in Arabidopsis, while Sultr1;1 plays a negligible role. Taken together, our study has identified a major transporter for Cr­(VI) uptake in plants, providing potential strategies for engineering plants with low Cr accumulation and consequently enhanced Cr­(VI) resistance and also plants with enhanced accumulation of Cr for the purpose of phytoremediation.