Squalene epoxidase (SQLE), a rate-limiting enzyme in cholesterol biosynthesis, is suggested as a proto-oncogene. Paradoxically, SQLE is degraded by excess cholesterol, and low SQLE is associated with aggressive colorectal cancer (CRC). Therefore, we studied the functional consequences of SQLE reduction in CRC progression.Gene and protein expression data and clinical features of CRCs were obtained from public databases and 293 human tissues, analyzed by immunohistochemistry. In-vitro studies demonstrated underlying mechanisms of CRC progression mediated by SQLE reduction. Mice were fed a 2% high-cholesterol or a control diet before and after cecum implantation of SQLE-genetically-knockdown/control CRC cells. Metastatic dissemination and circulating cancer stem cells were demonstrated by in-vivo tracking and flow cytometry analysis, respectively.In-vitro studies demonstrated that SQLE reduction helped cancer cells overcome constraints by inducing the epithelial-mesenchymal transition required to generate cancer stem cells. Surprisingly, SQLE interacted with GSK3β and p53. Active GSK3β contributes to the stability of SQLE, thereby increasing cell cholesterol content, whereas SQLE depletion disrupted the GSK3β/p53 complex, resulting in a metastatic phenotype. This was confirmed in a spontaneous CRC metastasis mice model where SQLE reduction, by a high-cholesterol regimen or genetic knockdown, strikingly promoted CRC aggressiveness through the production of migratory cancer stem cells.We demonstrated that SQLE reduction caused by cholesterol accumulation aggravates CRC progression via the activation of the β-catenin oncogenic pathway and deactivation of the p53 tumor suppressor pathway. Our findings provide new insights into the link between cholesterol and CRC, identifying SQLE as a key regulator in CRC aggressiveness and a prognostic biomarker.