Intracellular Ca2+ signals regulate insulin secretion from pancreatic β-cells. Although recent reports suggest that mitochondria influence insulin secretion through their Ca2+ uptake activity, mitochondrial Ca2+ dynamics in β-cells have remained elusive due to limitations in the method for mitochondrial Ca2+ visualization. Using recently developed high-performance intraorganellar Ca2+ indicators, CEPIA, we here analyzed high glucose-induced mitochondrial Ca2+ dynamics in an insulinoma cell line MIN6. Unexpectedly, high glucose-stimulation evoked only limited mitochondrial Ca2+ signals. Moreover, shRNA-mediated knockdown of mitochondrial calcium uptake 1 (MICU1), which is one of the essential regulators of mitochondrial Ca2+ uptake, significantly enhanced high glucose-induced mitochondrial Ca2+ signals. Furthermore, MICU1 knockdown caused a decrease in high glucose-induced insulin secretion. These results suggest that MICU1 negatively regulates mitochondrial Ca2+ uptake to maintain insulin secretion. Further analysis is required to clarify the relationship between mitochondrial Ca2+ uptake and insulin secretion.