The vacuolar H+-ATPases (V-ATPases) are essential, ATP-dependent proton pumps present in a variety of eukaryotic cellular membranes. Intracellularly, V-ATPase-dependent acidification functions in such processes as membrane traffic, protein degradation, autophagy and the coupled transport of small molecules. V-ATPases at the plasma membrane of certain specialized cells function in such processes as bone resorption, sperm maturation and urinary acidification. V-ATPases also function in disease processes such as pathogen entry and cancer cell invasiveness, while defects in V-ATPase genes are associated with disorders such as osteopetrosis, renal tubular acidosis and neurodegenerative diseases. This review highlights recent advances in our understanding of V-ATPase structure, mechanism, function and regulation, with an emphasis on the signaling pathways controlling V-ATPase assembly in mammalian cells. The role of V-ATPases in cancer and other human pathologies, and the prospects for therapeutic intervention, are also discussed.