Production of Aβ by γ-secretase is a key event in Alzheimer's disease (AD). The γ-secretase complex consists of presenilin (PS) 1 or 2, nicastrin (ncstn), Pen-2, and Aph-1 and cleaves type I transmembrane proteins, including the amyloid precursor protein (APP). Although ncstn is widely accepted as an essential component of the complex required for γ-secretase activity, recent in vitro studies have suggested that ncstn is dispensable for APP processing and Aβ production. The focus of this study was to answer this controversy and evaluate the role of ncstn in Aβ generation and the development of the amyloid-related phenotype in the mouse brain. To eliminate ncstn expression in the mouse brain, we used a ncstn conditional knockout mouse that we mated with an established AD transgenic mouse model (5XFAD) and a neuronal Cre-expressing transgenic mouse (CamKIIα-iCre), to generate AD mice (5XFAD/CamKIIα-iCre/ncstnf/f mice) where ncstn was conditionally inactivated in the brain. 5XFAD/CamKIIα-iCre/ncstnf/f mice at 10 week of age developed a neurodegenerative phenotype with a significant reduction in Aβ production and formation of Aβ aggregates and the absence of amyloid plaques. Inactivation of nctsn resulted in substantial accumulation of APP-CTFs and altered PS1 expression. These results reveal a key role for ncstn in modulating Aβ production and amyloid plaque formation in vivo and suggest ncstn as a target in AD therapeutics.