Muscle function is regulated by Ca2+, which mediates excitation-contraction coupling, energy metabolism, adaptation to exercise, and sarcolemmal repair. Several of these actions rely on Ca2+ delivery to the mitochondrial matrix via the mitochondrial Ca2+ uniporter, the pore of which is formed by mitochondrial calcium uniporter (MCU). MCU's gatekeeping and cooperative activation are controlled by MICU1. Loss-of-protein mutation in MICU1 causes a neuromuscular disease. To determine the mechanisms underlying the muscle impairments, we used MICU1 patient cells and skeletal muscle-specific MICU1 knockout mice. Both these models show a lower threshold for MCU-mediated Ca2+ uptake. Lack of MICU1 is associated with impaired mitochondrial Ca2+ uptake during excitation-contraction, aerobic metabolism impairment, muscle weakness, fatigue, and myofiber damage during physical activity. MICU1 deficit compromises mitochondrial Ca2+ uptake during sarcolemmal injury, which causes ineffective repair of the damaged myofibers. Thus, dysregulation of mitochondrial Ca2+ uptake hampers myofiber contractile function, likely through energy metabolism and membrane repair.