Mitochondrial F1FO ATP synthase is the key enzyme for mitochondrial bioenergetics. Dimeric F1FO-ATP synthase, is preferentially located at the edges of the cristae and its oligomerization state determines mitochondrial ultrastructure. The ATP synthase inhibitor protein IF1 modulates not only ATP synthase activity but also regulates both the structure and function of mitochondria. In order to understand this in more detail, we have investigated the effect of IF1 on the spatiotemporal organization of the ATP synthase. Stable cell lines were generated that overexpressed IF1 and constitutively active IF1-H49K. The expression of IF1-H49K induced a change in the localization and mobility of the ATP synthase as analyzed by single molecule tracking and localization microscopy (TALM). In addition, the ultrastructure and function of mitochondria in cells with higher levels of active IF1 displayed a gradual alteration. In state III, cristae structures were significantly altered. The inhibition of the hydrolase activity of the F1FO-ATP synthase by IF1 together with altered inner mitochondrial membrane caused re-localization and altered mobility of the enzyme.