Arabidopsis thaliana contains the putative K+ efflux transporters KEA1-KEA6, similar to KefB and KefC of Escherichia coli. KEA1-KEA3 are involved in the regulation of photosynthetic electron transport and chloroplast development. KEA4-KEA6 mediate pH regulation of the endomembrane network during salinity stress. However, the ion transport activities of KEA1-KEA6 have not been directly characterized. In this study, we used an E. coli expression system to examine KEA activity. KEA1-KEA3 and KEA5 showed bi-directional K+ transport activity, whereas KEA4 and KEA6 functioned as a K+ uptake system. The thylakoid membrane-localized Na+/H+ antiporter NhaS3 from the model cyanobacterium Synechocystis is the closest homolog of KEA3. Changing the putative Na+/H+ selective site of KEA3 (Gln-Asp) to that of NhaS3 (Asp-Asp) did not alter the ion selectivity without loss of K+ transport activity. The first residue in the conserved motif was not a determinant for K+ or Na+ selectivity. Deletion of the possible nucleotide-binding KTN domain from KEA3 lowered K+ transport activity, indicating that the KTN domain was important for this function. The KEA3-G422R mutation discovered in the Arabidopsis dpgr mutant increased K+ transport activity, consistent with the mutant phenotype. These results indicate that Arabidopsis KEA1-KEA6 act as K+ transport systems, and support the interpretation that KEA3 promotes dissipation of ΔpH in the thylakoid membrane.