Background: Alzheimer’s disease (AD) is characterized by the deposition of amyloid-β peptide (Aβ) in the brain. Aβ is produced by sequential β- and γ-secretase cleavages of amyloid precursor protein (APP). Clinical trials targeting β- and γ-secretases have all failed, partly because of the strong side effects. The aims of this work were to determine if the direct cleavage of APP by γ-secretase inhibits Aβ production, and to identify γ-cleavage-inhibiting signals within APP that can be targeted to prevent Aβ generation without inhibiting any enzyme. Methods: An APP mutant mimicking secreted APPγ was overexpressed in cells to test β-cleavage and Aβ production. APP deletion and truncation mutants were overexpressed in cells to identify the γ-secretase-inhibiting domain. The intracellular transport of the mutants was examined using immunofluorescence. Co-immunoprecipitation was performed to investigate the molecular mechanisms. Results: The APP N-terminal fragment mimicking the direct γ-cleavage product was not cleaved by beta-secretase 1 to produce detectable Aβ. However, in cells, the C-terminal fragments of APP longer than the last 116 residues could not be cleaved by γ-secretase in cells. No deletion mutant was cleaved by γ-secretase. C99, the direct precursor of Aβ, was no longer a γ-secretase substrate when green fluorescent protein was fused to its N-terminus. The large ectodomains prevented access to γ-secretase. Conclusions: Enabling the direct γ-cleavage of APP is a new and valid strategy to reduce Aβ. However, APP does not inhibit γ-cleavage via a specific inhibitory sequence in the ectodomain. Other methods to fulfill the strategy may benefit AD prevention and therapy