AbstractSTIM1 and Orai1 are the main components of a widely conserved Calcium influx pathway known as store-operated calcium entry (SOCE). STIM1 is a calcium sensor, which oligomerizes and activates Orai channels when calcium levels drop inside the endoplasmic reticulum (ER). The series of molecular rearrangements that STIM1 undergoes until final activation of Orai1 require the direct exposure of the STIM1 domain known as SOAR (Stim Orai Activating Region). In addition to these complex molecular rearrangements, other constituents like lipids at the plasma membrane, play critical roles orchestrating SOCE. PI(4,5)P2 and enriched cholesterol microdomains have been shown as important signaling platforms that recruit the SOCE machinery in steps previous to Orai1 activation. However, little is known about the molecular role of cholesterol once SOCE is activated. In this study we provide clear evidence that STIM1 has a cholesterol-binding domain located inside the SOAR region and modulates Orai1 channels. We demonstrate a functional association of STIM1 and SOAR to cholesterol, indicating a close proximity of SOAR to the inner layer of the plasma membrane. In contrast, the depletion of cholesterol induces the SOAR detachment from the plasma membrane and enhances its association to Orai1. These results are recapitulated with full length STIM1.