Plant vacuolar H+-transporting ATPases (V-ATPases) are ATP-driven proton pumps, located mainly in the vacuolar membrane, that generate proton motive force used to energize secondary active transports operating at the tonoplast. V-ATPases are the oldest and most complicated proton pumps found in plant cells. Enzyme subunits are divided into two major domains, the catalytic peripheral V1 domain responsible for ATP hydrolysis and the membrane-integral V domain responsible for H+ translocation. V sector consists of eight subunits named A-H whereas V complex is composed of a, c, c, d, and e subunits. In Arabidopsis thaliana, the 13 V-ATPase subunits are encoded by a total of 28 VHA genes suggesting the presence of individual subunit isoforms and different V-ATPase complexes specific to plant organs, tissues or physiological and developmental stages. The activity of plant V-ATPases is subjected to regulation at both the transcriptional and posttranslational levels. Potential mechanisms of biochemical regulation include reversible phosphorylation, redox regulation and modification induced by changes in the lipid composition of membrane. It has been postulated that under environmental stress the V-ATPase functions as a stress response enzyme, undergoing moderate changes in expression of subunits and modulation of enzyme activity. Since it was involved in ecophysiological adaptations at the molecular level, the V-ATPase was denominated an eco-enzyme. Numerous studies have confirmed an essential role of the V-ATPase in plant tolerance to environmental stresses including salinity, heavy metals and low temperature.