Vacuolar-type H+-adenosine triphosphatase (V-ATPase) is one of the most fundamental enzymes in nature. V-ATPases are responsible for the regulation of proton concentration in the intracellular acidic compartments. It has similar structure with the mitochondrial F0F1-ATP synthase (F-ATPase). dagger The V-ATPases are composed of multiple subunits and have various physiological functions, including membrane and organelle protein sorting, neurotransmitter uptake, cellular degradative processes, and cytosolic pH regulation. The V-ATPases have been involved in multidrug resistance. Recently, plasma membrane V-ATPases have been involved in regulation of extracellular acidity, essential for cellular invasiveness and proliferation in tumor metastasis. The current knowledge regarding the structure and function of V-ATPase and its role in cancer biology is discussed.