Mitochondrial homeostasis is crucial for the function of pancreatic beta-cells. ATP synthase inhibitory factor subunit 1 (IF1) is a mitochondrial protein interacting with ATP synthase to inhibit its enzyme activity. IF1 may also play a role in maintaining ATP synthase oligomerization and mitochondrial inner membrane formation. A recent study confirmed IF1 expresses in beta-cells. IF1 knockdown in cultured INS-1E beta-cells enhances glucose-induced insulin release. However, the role of IF1 in islet beta-cells remains little known. The present study investigates islets freshly isolated from mouse lines with global IF1 knockout (IF1(-/-)) and overexpression (OE). The glucose-stimulated insulin secretion was increased in islets from IF1(-/-) mice but decreased in islets from IF1 OE mice. Transmitted Electronic Microscopic assessment of isolated islets revealed that the number of matured insulin granules (with dense core) was relatively higher in IF1(-/-), but fewer in IF1 OE islets than those of controlled islets. The mitochondrial ultrastructure within beta-cells of IF1 overexpressed islets was comparable with those of wild-type mice, whereas those in IF1(-/-) beta-cells showed increased mitochondrial mass. Mitochondrial network analysis in cultured INS-1 beta-cells showed a similar pattern with an increased mitochondrial network in IF1 knockdown cells. IF1 overexpressed INS-1 beta-cells showed a compromised rate of mitochondrial oxidative phosphorylation with attenuated cellular ATP content. In contrast, INS-1 cells with IF1 knockdown showed markedly increased cellular respiration with improved ATP production. These results support that IF1 is a negative regulator of insulin production and secretion via inhibiting mitochondrial mass and respiration in beta-cells. Therefore, inhibiting IF1 to improve beta-cell function in patients can be a novel therapeutic strategy to treat diabetes. This report provides new evidence supporting the role of ATP synthase inhibitory factor subunit 1 (IF1), an endogenous ATP synthase inhibitory protein, in regulating beta-cell function. Investigations on genetic mouse models and an beta-cell line indicate that IF1 negatively regulates cellular respiration and mitochondrial homeostasis, thus controlling insulin storage and release from islets.