Abstract Prolactinoma is the most prevalent pituitary neuroendocrine tumor and dopamine agonists (DAs) targeting dopamine D2 receptor (DRD2) are recommended as the first-line treatment. However, varying degrees of DA resistance limit patient benefit. Our study used transcriptome sequencing of surgical tumor samples and found abnormal cholesterol metabolism in prolactinoma, especially in DA-resistant tumors. We found that cholesterol significantly enhanced the resistance of prolactinoma MMQ cell lines to cabergoline in vitro and in vivo xenografts. Further, cholesterol did not affect the total protein level of DRD2, but changed the distribution of DRD2 with downregulation of its membrane abundance and upregulation of cytoplasmic localization. Mechanistically, immunoprecipitation combined with mass spectrometry revealed cholesterol increased binding affinity between DRD2 and stress granules (SGs) core proteins, such as G3BP1. Western blot experiment of G3BP1 and fluorescent probe were used to confirm the formation of SGs after cholesterol treatment in MMQ cells and tumor xenografts, as well as in surgical tumor samples. Interfering the formation of SGs by overexpressing of USP10 and using the small molecule ISRIB reversed cholesterol’s effect on DRD2 cellular distribution and DA resistance in MMQ cells. Finally, a non-specificity inhibitor of SGs, anisomycin identified by drug repositioning analysis, could attenuate cholesterol-induced cabergoline resistance in vitro. Taken together, our findings suggest that abnormal cholesterol metabolism reduces DRD2 membrane localization via stress granules formation, which may be an important reason for the DA resistance of prolactinoma patients.