AbstractDuring muscle development, the sarco/endoplasmic reticulum (SR/ER) undergoes remodeling to establish a specialized internal Ca2+ store for muscle contraction. We hypothesized that store operated Ca2+ entry (SOCE) is required to fill Ca2+ stores and is, therefore, critical to creating a mature SR/ER. Stromal interaction molecule 1 (STIM1) functions as a sensor of internal Ca2+ store content and an activator of SOCE channels. Myocytes lacking STIM1 display reduced SR Ca2+ content and altered expression of key SR proteins. Sarcolipin (SLN), an inhibitor of the SR calcium pump, was markedly increased in the muscle of mutant STIM1 mice. SLN opposes the actions of STIM1 by limiting SOCE, reducing SR Ca2+ content and delaying muscle differentiation. During mouse muscle development SLN is highly expressed in embryonic muscle, while the expression of STIM1 is up‐regulated postnatally. These results suggest that SOCE regulates SR/ER specialization and that SLN and STIM1 act in opposing fashions to govern SOCE during myogenesis. Developmental Dynamics 241:639–647, 2012. © 2012 Wiley Periodicals, Inc.