For the past 20 years, the majority of cell culture studies reported that increasing cholesterol level increases amyloid-β (Aβ) production. Conversely, other studies and genetic evidences support that cellular cholesterol loss leads to Aβ generation. As a highly controversial issue in Alzheimer’s disease pathogenesis, the apparent contradiction prompted us to again explore the role of cellular cholesterol in Aβ production. Here, we adopted new neuronal and astrocytic cell models induced by 3β-hydroxysterol-Δ24 reductase (DHCR24), which obviously differ from the widely used cell models with overexpressing amyloid precursor protein (APP) in the majority of previous studies. In neuronal and astrocytic cell model, we found that deficiency of cellular cholesterol by DHCR24 knockdown obviously increased intracellular and extracellular Aβ generation. Importantly, in cell models with overexpressing APP, we found that APP overexpression could disrupt cellular cholesterol homeostasis and affect function of cells, coupled with the increase of APP β-cleavage product, 99-residue transmembrane C-terminal domain. Therefore, we suppose the results derived from the APP knockin models will need to be re-evaluated. One rational explanation for the discrepancy between our outcomes and the previous studies could be attributed to the two different cell models. Mechanistically, we showed that cellular cholesterol loss obviously altered APP intracellular localization by affecting cholesterol-related trafficking protein of APP. Therefore, our outcomes strongly support cellular cholesterol loss by DHCR24 knockdown leads to Aβ production.