In Arabidopsis, the nodulin 26-like intrinsic protein (NIP) subfamily of aquaporin proteins consists of nine members, five of which (NIP1;1, NIP1;2, NIP5;1, NIP6;1, and NIP7;1) were previously identified to be permeable to arsenite. However, the roles of NIPs in the root-to-shoot translocation of arsenite in plants remain poorly understood. In this study, using reverse genetic strategies, Arabidopsis NIP3;1 was identified to play an important role in both the arsenic uptake and root-to-shoot distribution under arsenite stress conditions. The nip3;1 loss-of-function mutants displayed obvious improvements in arsenite tolerance for aboveground growth and accumulated less arsenic in shoots than those of the wild-type plants, whereas the nip3;1 nip1;1 double mutant showed strong arsenite tolerance and improved growth of both roots and shoots under arsenite stress conditions. A promoter-β-glucuronidase analysis revealed that NIP3;1 was expressed almost exclusively in roots (with the exception of the root tips), and heterologous expression in the yeast Saccharomyces cerevisiae demonstrated that NIP3;1 was able to mediate arsenite transport. Taken together, our results suggest that NIP3;1 is involved in arsenite uptake and root-to-shoot translocation in Arabidopsis, probably as a passive and bidirectional arsenite transporter.