Abstract Aims/hypothesis Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca²⁺ uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca²⁺ importer (MCU) complex is thought to represent the main route for Ca²⁺ transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo. Methods Here, we inactivated the pore-forming subunit of the MCU, encoded by Mcu, selectively in mouse beta cells using Ins1^Cre-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca²⁺ concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance. Results Glucose-stimulated mitochondrial Ca²⁺ accumulation (p< 0.05), ATP production (p< 0.05) and insulin secretion (p< 0.01) were strongly inhibited in beta cell-specific Mcu-null (βMcu-KO) animals, in vitro, as compared with wild-type (WT) mice. Interestingly, cytosolic Ca²⁺ concentrations increased (p< 0.001), whereas mitochondrial membrane depolarisation improved in βMcu-KO animals. βMcu-KO mice displayed impaired in vivo insulin secretion at 5 min (p< 0.001) but not 15 min post-i.p. injection of glucose, whilst the opposite phenomenon was observed following an oral gavage at 5 min. Unexpectedly, glucose tolerance was improved (p< 0.05) in young βMcu-KO (<12 weeks), but not in older animals vs WT mice. Conclusions/interpretation MCU is crucial for mitochondrial Ca²⁺ uptake in pancreatic beta cells and is required for normal GSIS. The apparent compensatory mechanisms that maintain glucose tolerance in βMcu-KO mice remain to be established.