Cholesterol is a sterol lipid found in all higher eukaryotic organisms. It is required to consolidate the basic structural integrity and dynamic principles of cellular membranes and participates in many essential cellular processes that range from signal transduction to membrane traffic and metabolism. Moreover, a growing number of clinically highly relevant diseases such as immunological disorders or cancer has been linked to changes or misfunctions in cholesterol homeostasis. Therefore, the development of molecular tools that help to further unravel the role of cholesterol in essential cellular processes is of high relevance. Herein, we report the synthesis and proof-of-concept of a novel bifunctional imidazolium-based cholesterol analog (X-CHIM) that we envision to serve as a broadly applicable tool for the simultaneous investigation of cellular cholesterol distributions as well as cholesterol-protein interactions.