AbstractStromal interaction molecule 1 (STIM1) monitors ER-luminal Ca²⁺ levels to maintain cellular Ca²⁺ balance and to support Ca²⁺ signalling. The prevailing view has been that STIM1 senses reduced ER Ca²⁺ through dissociation of bound Ca²⁺ from a single EF-hand site, which triggers a dramatic loss of secondary structure and dimerization of the STIM1 luminal domain. Here we find that the STIM1 luminal domain has 5–6 Ca²⁺-binding sites, that binding at these sites is energetically coupled to binding at the EF-hand site, and that Ca²⁺ dissociation controls a switch to a second structured conformation of the luminal domain rather than protein unfolding. Importantly, the other luminal-domain Ca²⁺-binding sites interact with the EF-hand site to control physiological activation of STIM1 in cells. These findings fundamentally revise our understanding of physiological Ca²⁺ sensing by STIM1, and highlight molecular mechanisms that govern the Ca²⁺ threshold for activation and the steep Ca²⁺ concentration dependence.