Calcium ion (Ca2+) uptake into the mitochondrial matrix influences ATP production, Ca2+ homeostasis, and apoptosis regulation. Ca2+ uptake across the ion-impermeable inner mitochondrial membrane is mediated by the mitochondrial Ca2+ uniporter (MCU) complex. The MCU complex forms a pore structure composed of several proteins. MCU is a Ca2+-selective channel in the inner-mitochondrial membrane that allows electrophoretic Ca2+ entry into the matrix. Mitochondrial Ca2+ uptake 1 (MICU1) functions as a Ca2+-sensing regulator of the MCU complex. Previously, by microscopic analysis at the single-cell level, we found that during mast cell activation, mitochondria capture cytosolic Ca2+ in two steps. Consequently, mitochondrial Ca2+ uptake likely plays a role in cellular function through cytosolic Ca2+ buffering. Here, we investigate the role of MCU and MICU1 in mitochondrial Ca2+ uptake and mast cell degranulation using MCU- and MICU1-knockdown (KD) mast cells. Whereas MCU- and MICU1-KD mast cells show normal proliferation rates and mitochondrial membrane potential, they exhibit slow and reduced cytosolic and mitochondrial Ca2+ elevation after antigen stimulation. Moreover, beta-hexosaminidase release induced by antigen was significantly suppressed in MCU-KD cells but not MICU1-KD cells. This suggests that both MCU and MICU1 are involved in mitochondrial Ca2+ uptake in mast cells, while MCU plays a role in mast cell degranulation.