Membrane budding is an essential process during various biological activities, such as intercellular communication and transportation of life‐sustaining molecules and signals, which primarily occur with the aid of particular proteins. During such processes, the presence of cholesterol and its assembly into particular domains within the membranes have attracted considerable attention due to their unique characteristics in terms of the regulation of signal activities, membrane fluidity, and hormone production during metabolic pathways. However, despite these crucial roles, the precise mechanisms of their spatiotemporal localization toward specific areas and their physiological roles in membrane budding without the assistance of particular proteins remain unclear. Herein, a model membrane platform is reconstituted with lipid monolayer–bilayer junctions, which facilitate the selective concentration of cholesterol on the lipid monolayer regions. In vitro membrane budding is demonstrated by the remaining vesicles where the lipid monolayer membrane is ruptured. The physicochemical approach to the selective concentration of cholesterol and its consequent membrane vesiculation offer important clues to help understand the underlying mechanisms of cholesterol in the lipid‐driven membrane‐budding process.