Skeletal muscle is a highly-energy demanding tissue. ATP in muscle is derived from either anaerobic glycolysis or oxidative phosphorylation in mitochondria. As Ca2+ increases the activity of mitochondrial matrix dehydrogenases and the synthetic activity of the F1F0-ATPase, the rate of aerobic ATP generation is regulated by mitochondrial Ca2+. Mitochondrial Ca2+ uptake in non-excitable cells is determined by the mitochondrial Ca2+ uniporter (MCU). MCU forms homo-multimeric, Ca2+-permeable channels that coordinate mitochondrial Ca2+ entry upon Ca2+ release from the endoplasmic reticulum. Though still controversial, the mitochondrial leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) is proposed to be a Ca2+/H+ antiporter within the mitochondrial inner membrane. Here we determined the respective roles of MCU and LETM1 in activity dependent mitochondrial Ca2+ uptake in adult skeletal muscle. In vivo electroporation of MCU- and LETM1-specific siRNAs into mouse footpads resulted in significant knockdown (KD) of MCU (56±3%) and LETM1 (47±7%) expression in flexor digitorum brevis (FDB) muscles. To assess the impact of MCU and LETM1 on activity dependent mitochondrial Ca2+ uptake, we measured mitochondrial Ca2+ uptake following delivery of 5 successive high frequency tetanic stimulations (100Hz, 500ms, 0.2 duty cycle) in FDB fibers isolated from mice electroporated with either non-targeting control, MCU-, or LETM1 siRNAs. Mitochondrial Ca2+ uptake was reduced >50% in MCU KD FDB fibers compared to control (peak mitochondrial rhod-2 deltaF/F was 4.8±0.3 and 10.0±0.8, respectively). A similar reduction was observed following addition of 20 micromolar RU360, an MCU inhibitor (peak deltaF/F = 4.6±0.5). In addition, mitochondrial Ca2+ uptake during electrical stimulation was even more strongly inhibited (peak deltaF/F = 2.0±0.4) following transient over-expression of a dominant negative form of MCU (D260Q/E263Q). Activity-dependent mitochondrial Ca2+ uptake was not significantly altered following LETM1 knockdown. These results indicate that MCU function, but not LETM1 function, is required for optimal activity-dependent mitochondrial Ca2+ uptake in adult skeletal muscle fibers.