The vacuolar H(+)-ATPase (V-ATPase) is a universal component of eukaryotic organisms, which is present in both intracellular compartments and the plasma membrane. In the latter, its proton-pumping action creates the low intravacuolar pH, benefiting many processes such as, membrane trafficking, protein degradation, renal acidification, bone resorption, and tumor metastasis. In this article, we briefly summarize recent studies on the essential and diverse roles of mammalian V-ATPase and their medical applications, with a special emphasis on identification and use of V-ATPase inhibitors.