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Type 2C protein phosphatases (PP2Cs) are the largest protein phosphatase family. PP2Cs de-phosphorylate substrates for signaling in Arabi-dopsis, but the functions of most PP2Cs remain unknown. Here, we characterized PP2C49 (AT3G62260, a Group G PP2C), which regulates Na+distribution under salt stress and is localized to the cytoplasm and nucleus. PP2C49 was highly expressed in root vascular tissues and its dis-ruption enhanced plant tolerance to salt stress. Compared with wild type, the pp2c49 mutant contained more Na+in roots but less Na+in shoots and xylem sap, suggesting that PP2C49 regulates shoot Na+extrusion. Reciprocal grafting revealed a root-based mechanism underlying the salt toler-ance of pp2c49. Systemic Na+ distribution largely depends on AtHKT1;1 and loss of function of AtHKT1;1 in the pp2c49 background overrode the salt tolerance of pp2c49, resulting in salt sensitivity. Furthermore, compared with plants overexpressing PP2C49 in the wild-type background, plants over-expressing PP2C49 in the athtk1;1 mutant back-ground were sensitive to salt, like the athtk1;1 mutants. Moreover, protein–protein interaction and two-voltage clamping assays demonstrated that PP2C49 physically interacts with AtHKT1;1 and in-hibits the Na+permeability of AtHKT1;1. This study reveals that PP2C49 negatively regulates AtHKT1;1 activity and thus determines systemic Na+ alloca-tion during salt stress.