Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) is a key enzyme exclusively using free cholesterols as the substrates in cell and is involved in the cellular cholesterol homeostasis. In this study, we used human neuroblastoma cell line SK-N-SH as a model and first observed that inhibiting ACAT1 can decrease the amyloid precursor protein (APP)-α-processing. Meanwhile, the transfection experiments using the small interfering RNA and expression plasmid of ACAT1 indicated that ACAT1 can dependently affect the APP-α-processing. Furthermore, inhibiting ACAT1 was found to increase the free cholesterols in plasma membrane (PM-FC), and the increased PM-FC caused by inhibiting ACAT1 can lead to the decrease of the APP-α-processing, indicating that ACAT1 regulates the dynamics of PM-FC, which leads to the alteration of the APP-α-processing. More importantly, further results showed that under the ACAT1 inhibition, the alterations of the PM-FC and the subsequent APP-α-processing are not dependent on the cellular total cholesterol level, confirming that ACAT1 regulates the dynamics of PM-FC. Finally, we revealed that even when the Niemann–Pick-Type C-dependent pathway is blocked, the ACAT1 inhibition still obviously results in the PM-FC increase, suggesting that the ACAT1-dependent pathway is responsible for the shuttling of PM-FC to the intracellular pool. Our data provide a novel insight that ACAT1 which enzymatically regulates the dynamics of PM-FC may play important roles in the human neuronal cells.