Prorenin was long considered merely an inactive precursor of renin until the recent discovery of a specific (pro)renin receptor, (P)RR. The (P)RR non-proteolytically activates prorenin, resulting in Angiotensin I (Ang I) formation. The (P)RR, however, can also act as a bona fide signaling receptor, as binding of (pro)renin to the (P)RR can activate signaling pathways, independent of the generation of Ang I. The C-terminal domain of the (P)RR was previously identified as an accessory protein of vacuolar H+-ATPase (V-ATPase), and in collecting duct cells V-ATPase activity is required for (pro)renin induced ERK1/2 phosphorylation. Recent studies have also identified (pro)renin-independent functions for the (P)RR-V-ATPase axis in V-ATPase stability and Wnt signaling. However, so far the exact role of the (P)RR in the regulation of V-ATPase activity remains unknown. In this study, we knocked down the (P)RR in Mardin Darby canine kidney cells (MDCK.C11), that represent the intercalated cells of the collecting duct, using siRNA. We subsequently measured basal and vasopressin-induced V-ATPase activity in cells loaded with the pH-sensitive dye BCECF after an NH4Cl induced acid load. Transfection of MDCK.C11 cells with siRNA against (P)RR decreased (P)RR protein levels by 80%. Basal V-ATPase activity was similar in cells treated with siRNA targeting (P)RR (0.02905 ± 0.003934 pH units/min, n=23) and cells treated with control siRNA (0.03062 ± 0.003153 pH units/min, n=20). In the presence of 100 nM vasopressin, cells treated with control siRNA showed a significant (p<0.01) increase in V-ATPase activity (0.03565 ± 0.004543 pH units/min, n=24), whereas V-ATPase activity was not increased in cells treated with siRNA targeting (P)RR (0.02988 ± 0.004199 pH units/min, n=27). Western blotting showed decreased expression of the VOa2, but not the VOd2 subunit of the V-ATPase. Our results suggest that in collecting duct cells, the (P)RR is required for V-ATPase stability and vasopressin-induced V-ATPase activity, and indicate an important function for the (P)RR in V-ATPase regulation.