Recent studies have demonstrated the crucial role of cholesterol (Chol) in regulating the mechanical properties and biological functions of cell membranes. Methyl-β-cyclodextrin (MeβCD) is commonly utilized to modulate the Chol content in cell membranes, but there remains a lack of a comprehensive understanding. In this study, using a range of different techniques, we find that the optimal ratio of MeβCD to Chol for complete removal of Chol from a phosphocholine (PC)/Chol mixed membrane with a 1:1 mol ratio is 4.5:1, while the critical MeβCD-to-Chol ratio for membrane permeation falls within the range between 1.5 and 2.4. MeβCD at elevated concentrations induces the formation of fibrils or tubes from a PC membrane. Single lipid tracking reveals that removing Chol restores the diffusion of lipid molecules in the PC/Chol membrane to levels observed in pure PC membranes. Exposure to 5 mM MeβCD for 30 min effectively eliminates Chol from various cell lines, leading to an up to 8-fold enhancement in melittin cytotoxicity over Hela cells and an up to 3.5-fold augmentation of T cell cytotoxicity against B16F10-OVA cells. This study presents a diagram that delineates the concentration- and time-dependent distribution of MeβCD-induced Chol depletion and membrane deformation, which holds significant potential for modulating the mechanical properties of cellular membranes in prospective biomedical applications.