Objective 7-Dehydrocholesterol reductase (DHCR7) is an enzyme that plays a crucial regulatory role in sterol biosynthesis and has been implicated in tumorigenesis and progression. This study aims to elucidate the biological function of DHCR7 in the pathogenesis of colorectal cancer (CRC). Methods By integrating multi-omics data (including public genomic databases and mass spectrometry data from clinical samples) and establishing in vivo and in vitro experimental systems (encompassing animal models and CRC cell lines with gene overexpression and knockdown), we systematically investigated the functional role of DHCR7 in CRC. A multimodal research strategy combining bioinformatics analysis with molecular biology experiments (Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), western blotting, immunohistochemistry, etc .), proteomics analysis (liquid chromatography–mass spectrometry), and cellular functional assays (proliferation, apoptosis, migration, and invasion) was employed. Results Elevated levels of sterols were observed in CRC tumor tissues, and high cholesterol levels were found to promote the malignant phenotype of tumor cells. Mass spectrometry revealed that DHCR7 was significantly upregulated in CRC tissues and correlated with poor clinical prognosis. DHCR7 could modulate the cholesterol levels in CRC cells; overexpression of this gene enhanced cell proliferation, inhibited apoptosis, and promoted invasion and migration. Conversely, inhibition of DHCR7 expression abrogated these pro-tumorigenic effects, which was consistent with the inactivation of the PI3K/AKT/mTOR signaling pathway and confirmed by pathway reactivation experiments. DHCR7 deficiency significantly reduced tumorigenicity in vivo . Conclusion DHCR7 regulates the progression of CRC both in vitro and in vivo through the PI3K/AKT/mTOR signaling axis and affects the cholesterol levels in CRC.