The ubiquitous presence of stromal interaction molecule (STIM), endoplasmic reticulum (ER) Ca2+sensors, plays a crucial role for maintaining Ca2+homeostasis and signaling in mammalian cells by linking ER Ca2+depletion with extracellular Ca2+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 Ca2+affinities bothin celluloandin situ, alongside its impact on store-operated Ca2+entry (SOCE), we propose a novel mechanism wherein Glu II governs STIM1 activation by modulating Ca2+binding affinity, thereby adjusting the level of activated STIM1 in response to physiological stimulation.