Summary We combined the patch‐clamp technique with ratiometric fluorescence imaging using the proton‐responsive dye BCECF as a luminal probe. Upon application of a steep cytosol‐directed potassium ion (K+) gradient in Arabidopsis mesophyll vacuoles, a strong and reversible acidification of the vacuolar lumen was detected, whereas no associated electrical currents were observed, in agreement with electroneutral cation/H+ exchange. Our data show that this acidification was generated by NHX antiport activity, because: it did not distinguish between K+ and sodium (Na+) ions; it was sensitive to the NHX inhibitor benzamil; and it was completely absent in vacuoles from nhx1 nhx2 double knockout plants. Our data further show that NHX activity could be reversed, was voltage‐independent and specifically impaired by the low‐abundance signaling lipid PI(3,5)P2, which may regulate salt accumulation in plants by acting as a common messenger to coordinately shut down secondary active carriers responsible for cation and anion uptake inside the vacuole. Finally, we developed a theory based on thermodynamics, which supports the data obtained by our novel experimental approach. This work, therefore, represents a proof‐of‐principle that can be applied to the study of proton‐dependent exchangers from plants and animals, which are barely detectable using conventional techniques.