Amyloid-β (Aβ) is one of major molecules contributing to the pathogenesis of Alzheimer's disease (AD). Aβ is derived from amyloid-β protein precursor (AβPP) through sequential cleavages by β- and γ-secretases. Regulation of these components is thought to be an important factor in Aβ generation during the pathogenesis of AD. AβPP, β-secretase, and γ-secretase reside in lipid rafts, where cholesterol regulates the integrity and flexibility of membrane proteins and Aβ is generated. However, the relationship between cholesterol and Aβ generation is controversial. In this study, we aimed to elucidate the direct effects of cholesterol depletion on AβPP processing using AY9944, which blocks the last step of cholesterol biosynthesis and thus minimizes the unknown side effects of upstream inhibitors, such as HMG-CoA reductase inhibitors. Treatment with AY9944 decreased γ-secretase activity and Aβ generation. These results suggested that changes in membrane composition by lowering cholesterol with AY9944 affected γ-secretase activity and Aβ generation, which is associated with AD pathogenesis.