Vacuolar H⁺-ATPases are ubiquitous multisubunit complexes mediating the ATP-dependent transport of protons. In addition to their role in acidifying the lumen of various intracellular organelles, vacuolar H⁺-ATPases fulfill special tasks in the kidney. Vacuolar H⁺-ATPases are expressed in the plasma membrane in the kidney almost along the entire length of the nephron with apical and/or basolateral localization patterns. In the proximal tubule, a high number of vacuolar H⁺-ATPases are also found in endosomes, which are acidified by the pump. In addition, vacuolar H⁺-ATPases contribute to proximal tubular bicarbonate reabsorption. The importance in final urinary acidification along the collecting system is highlighted by monogenic defects in two subunits (ATP6V0A4, ATP6V1B1) of the vacuolar H⁺-ATPase in patients with distal renal tubular acidosis. The activity of vacuolar H⁺-ATPases is tightly regulated by a variety of factors such as the acid-base or electrolyte status. This regulation is at least in part mediated by various hormones and protein-protein interactions between regulatory proteins and multiple subunits of the pump.