Intracellular Ca²⁺ signal plays essential roles in insulin secretion from pancreatic β-cells. Although recent reports suggest that Ca²⁺ flows into the mitochondria via mitochondrial Ca²⁺ uniporter (MCU) is an important regulator of insulin secretion, mitochondrial Ca²⁺ dynamics in b-cells remain elusive due to limitations in the methods for the direct visualization analysis. Using recently developed high-performance intraorganellar Ca²⁺ indicator proteins, CEPIA, we here analyzed high glucose-induced mitochondrial Ca²⁺ dynamics in isolated mouse islets and  insulin-secreting MIN6 cells. Unexpectedly, high glucose stimulation failed to evoke a large amplitude of mitochondrial Ca²⁺ signals. shRNA-mediated knockdown of Micu1, which is one of the essential regulators of Ca²⁺ influx into the mitochondria, increased high glucose-induced mitochondrial Ca²⁺ signals. Micu1 knockdown caused significant decrease in insulin concentration after high glucose stimulation in MIN6 cells. Moreover, overexpression of MCU also increased mitochondrial Ca²⁺ signals and induced cell death. These results indicate that in b-cells, mitochondrial Ca²⁺ signals are tightly regulated by Micu1 during high glucose condition and aberrant regulation causes pancreatic β-cell dysfunction.