The role of the mitochondrial Ca²⁺ uniporter (MCU) in physiologic cell proliferation remains to be defined. Here, we demonstrated that the MCU was required to match mitochondrial function to metabolic demands during the cell cycle. During the G₁-S transition (the cycle phase with the highest mitochondrial ATP output), mitochondrial fusion, oxygen consumption, and Ca²⁺ uptake increased in wild-type cells but not in cells lacking MCU. In proliferating wild-type control cells, the addition of the growth factors promoted the activation of the Ca²⁺/calmodulin-dependent kinase II (CaMKII) and the phosphorylation of the mitochondrial fission factor Drp1 at Ser⁶¹⁶. The lack of the MCU was associated with baseline activation of CaMKII, mitochondrial fragmentation due to increased Drp1 phosphorylation, and impaired mitochondrial respiration and glycolysis. The mitochondrial fission/fusion ratio and proliferation in MCU-deficient cells recovered after MCU restoration or inhibition of mitochondrial fragmentation or of CaMKII in the cytosol. Our data highlight a key function for the MCU in mitochondrial adaptation to the metabolic demands during cell cycle progression. Cytosolic CaMKII and the MCU participate in a regulatory circuit, whereby mitochondrial Ca²⁺ uptake affects cell proliferation through Drp1.