The vacuolar (H+)-ATPase (V-ATPase) is a universal proton pump and its activity is required for a variety of cell-biological processes such as membrane trafficking, receptor-mediated endocytosis, lysosomal protein degradation, osteoclastic bone resorption and maintenance of acid-base homeostasis by renal intercalated cells. In neuronal and neuroendocrine cells, the V-ATPase is the major regulator of intragranular acidification which is indispensable for correct prohormone processing and neurotransmitter uptake. In these specialized cells, the V-ATPase is equipped with the accessory subunits ATP6AP1/Ac45 and ATP6AP2/(pro) renin receptor. Recent studies have shown that Ac45 interacts with the V0- sector of the V-ATPase complex, thereby regulating the intragranular pH and Ca2+-dependent exocytotic membrane fusion. Thus, Ac45 can be considered as a V-ATPase regulator in the neuroendocrine secretory pathway. ATP6AP2 has recently been found to be identical to the (pro) renin receptor and has a dual role: (i) in the renin-angiotensin system that also regulates V-ATPase activity; (ii) acting as an adapter by binding to both the V-ATPase and the Wnt receptor complex, thereby recruiting the receptor complex into an acidic microenvironment. We here provide an overview of the two V-ATPase accessory subunits as novel key players in V-ATPase regulation. We argue that the accessory subunits are candidate genes for V-ATPase-related human disorders and promising targets for manipulating V-ATPase functioning in vivo.