Reprogramming of lipid metabolism is an important hall mark of cancer cells that is linked to therapy resistance. Through spatiotemporally resolved quantitative lipid imaging, we have discovered that site-specifically enriched cholesterol in cancer cells orchestrates complex cell-autonomous oncogenic signaling networks that constitutively drive cell stemness and resistance to cell death. Proximity labeling analysis identified a number of kinases, phosphatases, and scaffolding proteins that are directly and specifically controlled by cholesterol. Small molecule inhibitors specifically targeting cholesterol-protein interaction serve as potent and specific cancer type-specific therapeutic agents with novel pharmacological properties, including suppression of cancer cell stemness and drug resistance.