Significance Cholesterol regulates critical cell functions, including lysis, viral budding, and antibiotic resistance, by modifying the bending rigidity of cell membranes; i.e., the ability of membranes to bend or withstand mechanical stresses. A molecular-level understanding of these functions requires knowledge of how cholesterol modifies membrane mechanics over relevant length and time scales. Currently, it is widely accepted that cholesterol has no effect on the mechanical properties of unsaturated lipid membranes, implying that viruses, for example, can bud from regions enriched in (poly)unsaturated lipids. Our observations that cholesterol causes local stiffening in DOPC membranes indicate that a reassessment of existing concepts is necessary. These findings have far-reaching implications in understanding cholesterol’s role in biology and its applications in bioengineering and drug design.