We investigated the role of three uncharacterized cation transporters of Pseudomonas aeruginosa PAO1 as virulence factors for barley: PA1207, PA5021, and PA2647. PAO1 displayed reduced barley virulence with inactivated PA1207, PA5021, and PA2647 as well as with one known Na+/H+ antiporter, PA1820. Using the Escherichia coli LB2003 mutant lacking three K+ uptake systems, the expression of the PA5021 gene repressed LB2003 growth with low K+, but the strain acquired tolerance to high K+. In contrast, the expression of the PA1207 gene enhanced growth of LB2003 with low K+ but repressed its growth with high K+; therefore, the PA5021 protein exports K+, while the PA1207 protein imports K+. The PA5021 mutant of P. aeruginosa also showed impaired growth at 400 mM KCl and at 400 mM NaCl; therefore, the PA5021 protein may also export Na+. The loss of the PA5021 protein also decreased production of the virulence factor pyocyanin; corroborating this result, pyocyanin production decreased in wild-type PAO1 under high salinity. Whole-genome transcriptome analysis showed that PAO1 induced more genes in barley upon infection compared to the PA5021 mutant. Additionally, PAO1 infection induced water stress-related genes in barley, which suggests that barley may undergo water deficit upon infection by this pathogen.