Store-operated Ca2 entry (SOCE) is a major Ca2 signaling pathway facilitating extracellular Ca2 influx in response to the initial release of intracellular endo/sarcoplasmic reticulum (ER/ SR) Ca2 stores. Stromal interaction molecule 1 (STIM1) is the Ca2 sensor that activates SOCE following ER/SR Ca2 depletion. The EF-hand and the adjacent sterile -motif (EFSAM) domains of STIM1 are essential for detecting changes in luminal Ca2 concentrations. Low ER Ca2 levels trigger STIM1 destabilization and oligomerization, culminating in the opening of Orai1-composed Ca2 channels on the plasma membrane. NO-mediated S-nitrosylation of cysteine thiols regulates myriad protein functions, but its effects on the structural mechanisms that regulate SOCE are unclear. Here, we demonstrate that S-ni-trosylation of Cys49 and Cys56 in STIM1 enhances the thermodynamic stability of its luminal domain, resulting in suppressed hydrophobic exposure and diminished Ca2 depletion– dependent oligomerization. Using solution NMR spectroscopy, we pinpointed a structural mechanism for STIM1 stabilization driven by complementary charge interactions between an electropositive patch on the core EFSAM domain and the S-nitrosy-lated nonconserved region of STIM1. Finally, using live cells, we found that the enhanced luminal domain stability conferred by either Cys49 and Cys56 S-nitrosylation or incorporation of negatively charged residues into the EFSAM electropositive patch in the full-length STIM1 context significantly suppresses SOCE. Collectively, our results suggest that S-nitrosylation of STIM1 inhibits SOCE by interacting with an electropositive patch on the EFSAM core, which modulates the thermodynamic stability of the STIM1 luminal domain.