Updated project metadata. Oxysterols (OHCs) are hydroxylated cholesterol metabolites that play ubiquitous roles in health and disease. Due to the non-covalent nature of their interactions and unique partitioning in membranes, the analysis of live-cell, proteome-wide interactions of OHCs remains an unmet challenge. In this Resource, we present a structurally precise chemoproteomics probe for the osteogenic molecule 20(S)-hydroxycholesterol (20(S)-OHC) and provide a map of its proteome-wide targets in the membranes of living cells. Our target catalogue consolidates diverse OHC ontologies and demonstrates that OHC-interacting proteins cluster with specific processes in immune response and cancer. Competition experiments reveal that 20(S)-OHC is a chemo-, regio-, and stereoselective ligand for the protein Tmem97 (σ2 receptor), enabling molecular reconstruction of the Tmem97:20(S)-OHC binding site. Our results demonstrate that multiplexed, quantitative analysis of cellular target engagement can expose new dimensions of OHC activity and identify actionable targets for molecular therapy.