F(o)F(1)-ATP synthase (F(o)F(1)) synthesizes ATP in mitochondria coupled with proton flow driven by the proton motive force (pmf) across membranes. It has been known that isolated IF1, an evolutionarily well conserved mitochondrial protein, can inhibit the ATP hydrolysis activity of F(o)F(1). Here, we generated HeLa cells with permanent IF1 knockdown (IF1-KD cells) and compared their energy metabolism with control cells. Under optimum growth conditions, IF1-KD cells have lower cellular ATP levels and generate a higher pmf and more reactive oxygen species. Nonetheless, IF1-KD cells and control cells show the same rates of cell growth, glucose consumption, and mitochondrial ATP synthesis. Furthermore, contrary to previous reports, the morphology of mitochondria in IF1-KD cells appears to be normal. When cells encounter sudden dissipation of pmf, the cytoplasmic ATP level in IF1-KD cells drops immediately (~1 min), whereas it remains unchanged in the control cells, indicating occurrence of futile ATP hydrolysis by F(o)F(1) in the absence of IF1. The lowered ATP level in IF1-KD cells then recovers gradually (~10 min) to the original level by consuming more glucose than control cells. The viability of IF1-KD cells and control cells is the same in the absence of pmf. Thus, IF1 contributes to ATP homeostasis, but its deficiency does not affect the growth and survival of HeLa cells. Only when cells are exposed to chemical ischemia (no glycolysis and no respiration) or high concentrations of reactive oxygen species does IF1 exhibit its ability to alleviate cell injury.