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Specialized junctional sites that connect the plasma membrane (PM) and endoplasmic reticulum (ER) play critical roles in controlling lipid metabolism and Ca²⁺ signalling^,,,. Store-operated Ca²⁺ entry mediated by dynamic STIM1–ORAI1 coupling represents a classical molecular event occurring at ER–PM junctions, but the protein composition and how previously unrecognized protein regulators facilitate this process remain ill-defined. Using a combination of spatially restricted biotin labelling in situ coupled with mass spectrometry^, and a secondary screen based on bimolecular fluorescence complementation, we mapped the proteome of intact ER–PM junctions in living cells without disrupting their architectural integrity. Our approaches led to the discovery of an ER-resident multi-transmembrane protein that we call STIMATE (STIM-activating enhancer, encoded by TMEM110) as a positive regulator of Ca²⁺ influx …