Limb-Girdle Muscular Dystrophy R1/2A (LGMD2A) is an autosomal recessive muscle disease characterized by progressive weakness of pelvic and scapular muscles. LGMD2A results from mutations in Calpain-3 (CAPN3), a calcium-dependent, non-lysosomal protease strictly expressed in skeletal muscle. Although LGMD2A is the most prevalent LGMD, the exact contribution of CAPN3 loss to the variable pathology remains unclear. Initially, CAPN3 was found to maintain sarcomere integrity by regulating sarcomere remodeling through its localization on Titin. However, there is evidence for CAPN3 localization at the triad, thus it may contribute to Ca2+ regulation during excitation-contraction coupling. In particular, sarco/endo-plasmic reticulum Ca2+ ATPase (SERCA) activity is significantly reduced in calpain-3 knockout (C3KO) mice, which may lead to SR store depletion and the activation of store operated Ca2+ entry (SOCE) to replenish the SR Ca2+ store. SOCE activity was assessed in resting muscles then subsequently evoked by treadmill running in C57BL/6J (C57) and C3KO 6-month-old male mice. Fiber bundles from Extensor Digitorum Longus (EDL) muscles were analyzed using immunohistochemistry for proteins associated with SOCE activity. SOCE activity was also measured in Flexor Digitorum Brevis (FDB) fibers via Indo-1 Ca2+ measurements. Surprisingly, muscles from C3KO mice showed evidence of high and variable SOCE activity at rest, which diminished following treadmill running. In contrast, muscles from C57 mice had modest SOCE at rest, which was elevated after treadmill running. This was substantiated by greater susceptibility to fatigue post-treadmill running in EDL muscles from C3KO mice, aberrant localization of SOCE proteins in EDL fiber bundles from C3KO mice, and reduced Ca2+ release in C3KO FDB fibers 1 hr post-treadmill running. Thus, constitutively active and aberrant SOCE response to exercise in C3KO muscles may represent a newly identified mechanism underlying pathology of LGMD2A and supports a role of CAPN3 in Ca2+ homeostasis and handling.