Cholesterol is ubiquitous in biology, shaping membrane properties and serving as a biosynthetic precursor for essential signaling molecules and hormones─and, in some contexts, acting as a signaling molecule itself. Here, we describe the development of photoswitchable versions of cholesterol that retain the lipophilic profile of the parent compound. These analogs were designed through computationally guided replacement of the native iso-octyl side chain with azobenzene-based photoswitches. Our compounds, termed photocholesterols (PChols), were assembled through a modular and readily diversifiable semisynthetic route involving transition metal-catalyzed cross-couplings followed by stereo- and chemoselective hydrogenations. They can be used to optically control binding to the sterol transport proteins ORP1/2 and OSBP, which play key roles in distributing cholesterol within intracellular compartments. Our work establishes a template for photoresponsive sterols that closely mimic cholesterol and may be applied broadly to investigate cholesterol’s roles in membrane behavior, signaling, and transport.