ABSTRACTExtensive research has focused on cellular cholesterol and its regulation, primarily due to its crucial physiological roles and its association with numerous diseases resulting from dysregulated homeostasis. Consequently, investigating cholesterol metabolism and the network of regulating proteins remains an ongoing challenge for biomedical research seeking new molecular targets to manage aberrant cholesterol levels in pathologic conditions. There is evidence that Sigma‐2/TMEM97 receptor regulates cholesterol metabolism. However, the mechanisms remain incompletely understood to date. Therefore, this study aimed to employ a pharmacological approach based on selective Sigma‐2/TMEM97 agonists, rimcazole and siramesine, to uncover the contribution of this receptor to cholesterol homeostasis. Our results indicate that Sigma‐2/TMEM97 activation modulates cholesterol uptake by altering key proteins involved in, leading to free cholesterol and neutral lipids accumulation. This sheds light on potential mechanisms implied, contributing a new piece to the intricate puzzle of cholesterol metabolism homeostasis.