Recently, we found that vacuolar proton ATPase (VPATPase) operates in cardiomyocytes as a complementary proton-extruding mechanism. Its activity was increased by preconditioning with resultant attenuation of intracellular acidification during ischemia. In this study, we examined whether VPATPase-mediated proton efflux during metabolic inhibition/recovery may spare Na+ overload via Na+-H+ exchange, attenuate Na+-Ca2+ exchange, and decrease apoptosis. Neonatal rat cardiomyocytes were subjected to 2- to 3-hour metabolic inhibition with cyanide and 2-deoxyglucose and 24-hour recovery. The effect of VPATPase inhibition by 50 nmol/L bafilomycin A1 on apoptosis, pHi, and [Ca2+]i was studied by flow cytometry with propidium iodide, seminaphthorhodafluor (SNARF)-1-AM, and indo-1-AM staining, respectively. VPATPase inhibition increased the amount of apoptosis measured after 24 hours of recovery and abrogated the protective effect of inhibition of Na+-H+ exchange by (5-N-ethyl-N-isopropyl)amiloride (EIPA). Dual blockade of VPATPase and Na+-H+ exchange was additive in effect with EIPA on pHi during metabolic inhibition/recovery and recovery from the acid challenge with sodium propionate. VPATPase blockade increased the rate of accumulation of intracellular Ca2+ at the beginning of metabolic inhibition and abrogated the delaying effect of EIPA on intracellular Ca2+ accumulation. These results indicate that VPATPase plays an important accessory role in cardiomyocyte protection by reducing acidosis and Na+-H+ exchange-induced Ca2+ overload.