Stromal interaction molecule-1 (STIM1) activates store-operated Ca²⁺ entry (SOCE) in response to diminished luminal Ca²⁺ levels. Here, we present the atomic structure of the Ca²⁺-sensing region of STIM1 consisting of the EF-hand and sterile α motif (SAM) domains (EF-SAM). The canonical EF-hand is paired with a previously unidentified EF-hand. Together, the EF-hand pair mediates mutually indispensable hydrophobic interactions between the EF-hand and SAM domains. Structurally critical mutations in the canonical EF-hand, "hidden" EF-hand, or SAM domain disrupt Ca²⁺ sensitivity in oligomerization via destabilization of the entire EF-SAM entity. In mammalian cells, EF-SAM destabilization mutations within full-length STIM1 induce punctae formation and activate SOCE independent of luminal Ca²⁺. We provide atomic resolution insight into the molecular basis for STIM1-mediated SOCE initiation and show that the folded/unfolded state of the Ca²⁺-sensing region of STIM is crucial to SOCE regulation.