Alzheimer's disease (AD) represents the most common age-related neurodegenerative disorder. It is characterized by the invariant accumulation of the β-amyloid peptide (Aβ), which mediates synapse loss and cognitive impairment in AD. Current therapeutic approaches concentrate on reducing Aβ levels and amyloid plaque load via modifying or inhibiting the generation of Aβ. Based on in vivo two-photon imaging, we present evidence that side effects on the level of dendritic spines may counteract the beneficial potential of these approaches. Two potent γ-secretase inhibitors (GSIs), DAPT (N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester) and LY450139 (hydroxylvaleryl monobenzocaprolactam), were found to reduce the density of dendritic spines in wild-type mice. In mice deficient for the amyloid precursor protein (APP), both GSIs had no effect on dendritic spine density, demonstrating that γ-secretase inhibition decreases dendritic spine density via APP. Independent of the effects of γ-secretase inhibition, we observed a twofold higher density of dendritic spines in the cerebral cortex of adult APP-deficient mice. This observation further supports the notion that APP is involved in the modulation of dendritic spine density—shown here for the first time in vivo.