AbstractStromal interaction molecule−1 and −2 (STIM1/2) are endoplasmic reticulum (ER) membrane-inserted calcium (Ca²⁺) sensing proteins that, together with Orai1-composed Ca²⁺ channels on the plasma membrane (PM), regulate intracellular Ca²⁺ levels. Recent evidence suggests that S-nitrosylation of the luminal STIM1 Cys residues inhibits store operated Ca²⁺ entry (SOCE). However, the effects of thiol modifications on STIM2 during nitrosative stress and their role in regulating basal Ca²⁺ levels remain unknown. Here, we demonstrate that the nitric oxide (NO) donor nitrosoglutathione (GSNO) thermodynamically stabilizes the STIM2 Ca²⁺ sensing region in a Cys-specific manner. We uncovered a remarkable synergism in this stabilization involving the three luminal Cys of STIM2, which is unique to this paralog. S-Nitrosylation causes structural perturbations that converge on the face of the EF-hand and sterile α motif (EF-SAM) domain, implicated in unfolding-coupled activation. In HEK293T cells, enhanced free basal cytosolic Ca²⁺ and SOCE mediated by STIM2 overexpression could be attenuated by GSNO or mutation of the modifiable Cys located in the luminal domain. Collectively, we identify the Cys residues within the N-terminal region of STIM2 as modifiable targets during nitrosative stress that can profoundly and cooperatively affect basal Ca²⁺ and SOCE regulation.