Mechanisms that regulate water channels in the plant plasma membrane (PM) were investigated in Arabidopsis suspension cells. Cell hydraulic conductivity was measured with a cell pressure probe and was reduced 4-fold as compared to control values when calcium was added in the pipette and in bathing solution. To assess the significance of these effects in vitro, PM vesicles were isolated by aqueous two-phase partitioning and their water transport properties were characterized by stopped-flow spectrophotometry. Membrane vesicles isolated in standard conditions exhibited reduced water permeability (Pf) together with a lack of active water channels. In contrast, when prepared in the presence of chelators of divalent cations, PM vesicles showed a 2.3-fold higher Pf and active water channels. Furthermore, equilibration of purified PM vesicles with divalent cations reduced their Pf and water channel activity down to the basal level of membranes isolated in standard conditions. Ca2+ was the most efficient with a half-inhibition of Pf at 50–100 µm free Ca2+. Water transport in purified PM vesicles was also reversibly blocked by H+, with a half-inhibition of Pf at pH 7.2–7.5. Thus, both Ca2+ and H+ contribute to a membrane-delimited switch from active to inactive water channels that may allow coupling of water transport to cell signalling and metabolism.