Cholesterol has been shown to be a potential risk factor for the occurrence and progression of breast cancer. This study aimed to investigate the regulation of DHCR7 in cholesterol synthesis and its role in Hedgehog (Hh) signaling pathway activation, as well as its impact on the progression of triple-negative breast cancer (TNBC).We analyzed the gene expression data from the GSE76275 data set by bioinformatics analysis to determine the expression of cholesterol-related genes in triple-negative breast cancer. In the triple-negative breast cancer cell lines, including BT-549 and MDA-MB-231, RNA interference gene knockout was used to evaluate the functional impact of DHCR7. In addition, the SMO mutant (SMOV329F) with anti-cholesterol binding inhibition was introduced to determine its interaction with the pathway changes mediated by DHCR7. Cell proliferation, migration, and signaling pathway activation were assessed through Western blotting, CCK-8 assay, transwell migration assay, and qPCR.DHCR7 expression was significantly elevated in TNBC tissues and cell lines, enhancing the Hh pathway activity through cholesterol modulation. Knocking down DHCR7 and the SMOV329F mutation both reduced the expression of Hedgehog-related proteins and inhibited cell proliferation and migration abilities. However, the SMOV329F mutation re-versed the inhibitory effect of knocking down DHCR7 on TNBC cells.DHCR7 activates the Hedgehog pathway by regulating cholesterol to promote the development of TNBC. These findings provide insights into the regulatory roles of DHCR7 in cholesterol-related pathways and Hh signaling in TNBC cells, offering potential therapeutic targets for TNBC treatment.Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.