The primary cilium is a specialized signaling hub whose function depends on a tightly regulated membrane composition. While its protein content is well-characterized, its lipid identity, particularly regarding sterols, remains poorly defined. Here, we used mass spectrometry-based lipidomics to map the sterol profile of isolated primary cilia from MDCK cells. We found that ciliary membranes are enriched in cholesterol and desmosterol while excluding precursors like 7-lathosterol and limiting others, suggesting a selective sterol barrier. Inhibiting cholesterol biosynthesis at distinct enzymatic steps led to sterol accumulation, altered ciliary membrane fluidity, and impaired Hedgehog signaling, including defective Smoothened (Smo) retention-even in the presence of a constitutively active form of Smo. These findings link sterol homeostasis to ciliary membrane properties and signaling fidelity. Our work provides a molecular framework for understanding Hedgehog-related phenotypes in disorders like Smith-Lemli-Opitz Syndrome, highlighting the importance of membrane lipid composition in developmental signaling.