The ubiquitous presence of stromal interaction molecule (STIM), endoplasmic reticulum (ER) Ca 2+ sensors, plays a crucial role for maintaining Ca 2+ homeostasis and signaling in mammalian cells by linking ER Ca 2+ depletion with extracellular Ca 2+ influx. Although recent advancements have shed light on glycosylation in STIM proteins, further exploration of deglycosylation enzyme modification and its role remains limited by methodological constraints. In this study, leveraging the miniTurbo-driven proximal biotinylation labeling method, we were able to screen for weak and transiently binding protein molecules within specific environments like ER lumina. Herein, we unveil glucosidase II, comprising GANAB and PRKCSH, as a novel partner of the STIM1 complex. Through investigations into Glucosidase II’s (Glu II) regulation of STIM1’s Ca 2+ affinities both in cellulo and in situ , alongside its impact on store-operated Ca 2+ entry (SOCE), we propose a novel mechanism wherein Glu II governs STIM1 activation by modulating Ca 2+ binding affinity, thereby adjusting the level of activated STIM1 in response to physiological stimulation.