Our recent work identified Store-Operated Ca2+ Entry (SOCE) as the critical Ca2+-source required for the induction of human myoblast differentiation (1). The present work indicates that STIM2 silencing, as STIM1 silencing, reduces myoblast SOCE amplitude and differentiation. As myoblasts in culture can be induced to differentiate into myotubes, which spontaneously contract in culture, we used the same molecular tools to explore whether the Ca2+-mechanism of excitation-contraction coupling also relies on STIM1 and STIM2. Live cell imaging of early differentiating myoblasts revealed a characteristic clustering of activated STIM1 and STIM2 during the first few hours of differentiation. Thapsigargin-induced depletion of endoplasmic reticulum Ca2+ content caused STIM1 and STIM2 redistribution into clusters, and co localization of both STIM proteins. Interaction of STIM1 and STIM2 was revealed by a rapid increase in FRET between CFP-STIM1 and YFP-STIM2 after SOCE activation and confirmed by co immunoprecipitation of endogenous STIM1 and STIM2. Although both STIM proteins clearly contribute to SOCE and are required during the differentiation process, STIM1 and STIM2 are functionally largely redundant as over-expression of either STIM1 or STIM2 corrected most of the impact of STIM2 or STIM1 silencing on SOCE and differentiation. With respect to excitation-contraction, we observed that human myotubes rely also on STIM1 and STIM2 to refill their endoplasmic reticulum Ca2+-content during repeated KCl induced Ca2+ releases. This indicates that STIM2 is a necessary partner of STIM1 for excitation/contraction coupling. Thus, both STIM proteins are required and interact to control SOCE during human myoblast differentiation and human myotubes excitation/contraction coupling.