Store-operated Ca²⁺ entry (SOCE), a ubiquitous mechanism that allows recovery of Ca²⁺ ions from the extracellular space, has been proposed to limit fatigue during repetitive skeletal muscle activity. However, the subcellular location for SOCE in muscle fibers has not been unequivocally identified. Here we show that exercise drives a significant remodeling of the sarcotubular system to form previously unidentified junctions between the sarcoplasmic reticulum (SR) and transverse-tubules (TTs). We also demonstrate that these new SR-TT junctions contain the molecular machinery that mediate SOCE: stromal interaction molecule-1 (STIM1), which functions as the SR Ca²⁺ sensor, and Orai1, the Ca²⁺-permeable channel in the TT. In addition, EDL muscles isolated from exercised mice exhibit an increased capability of maintaining contractile force during repetitive stimulation in the presence of 2.5 mM extracellular Ca²⁺, compared to muscles from control mice. This functional difference is significantly reduced by either replacement of extracellular Ca²⁺ with Mg²⁺ or the addition of SOCE inhibitors (BTP-2 and 2-APB). We propose that the new SR-TT junctions formed during exercise, and that contain STIM1 and Orai1, function as Ca²⁺Entry Units (CEUs), structures that provide a pathway to rapidly recover Ca²⁺ ions from the extracellular space during repetitive muscle activity.