Neuronal activity has an impact on β cleavage of amyloid precursor protein (APP) by BACE1 to generate amyloid-β peptide (Aβ). However, the molecular mechanisms underlying this effect remain to be elucidated. Cholesterol dependency of β cleavage prompted us to analyze immunoisolated APP-containing detergent-resistant membranes from rodent brains. We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains. In living cells, APP associates with syntaxin 1–containing microdomains through X11–Munc18, which inhibits the APP–BACE1 interaction and β cleavage via microdomain segregation. Phosphorylation of Munc18 by cdk5 causes a shift of APP to BACE1-containing microdomains. Neuronal hyperactivity, implicated in Aβ overproduction, promotes the switching of APP microdomain association as well as β cleavage in a partially cdk5-dependent manner. We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons.