Intracellular Ca²⁺ signaling regulates cell migration by acting on cytoskeleton architecture, cell directionality and focal adhesions dynamics. In migrating cells, cytosolic Ca²⁺ pool and Ca²⁺ pulses are described as key components of these effects. Whereas the role of the mitochondrial calcium homeostasis and the Mitochondria Cacium Uniporter (MCU) in cell migration were recently highlighted in vivo using the zebrafish model, their implication in actin cystokeleton dynamics and cell migration in mammals is not totally characterized. Here, we show that mcu silencing in two human cell lines compromises their migration capacities. This phenotype is characterized by actin cytoskeleton stiffness, a cell polarization loss and an impairment of the focal adhesion proteins dynamics. At the molecular level, these effects appear to be mediated by the reduction of the ER and cytosolic Ca²⁺ pools, which leads to a decrease in Rho-GTPases, RhoA and Rac1, and Ca²⁺-dependent Calpain activites, but seem to be independent of intracellular ATP levels. Together, this study highlights the fundamental and evolutionary conserved role of the mitochondrial Ca²⁺ homeostasis in cytoskeleton dynamics and cell migration.