Depletion of ATP is a promising strategy for controlling or killing pests, pathogens, and cancer cells. In eukaryotic cells, mitochondrial F₁F_o-ATP synthase plays a central role in oxidative phosphorylation and has therefore been considered as a suitable target for this strategy against pests, pathogenic fungi, and some cancer cells. Membrane-embedded Atp9 (subunit c) is a crucial subunit of the F₁F_o-ATP synthase ring oligomer (c-ring) that, together with Atp6, directly mediates proton translocation, which drives ATP synthesis. Substitution of the proton-binding residue of Atp9, i.e., glutamic acid, with glutamine abrogates ATP synthase function. Importantly, a single mutated Atp9 subunit in the c-ring is sufficient for inactivation. We hypothesized, therefore, that heterologous expression of mutant Atp9 in cells would result in the assembly of a nonfunctional complex and, consequently, depletion of ATP. Using a yeast model system, we verified the hypothesis through a series of biochemical analyses and thus validated this approach as a strategy for depleting intracellular ATP in target cells and organisms.