The γ-secretase complex, composed of presenilin, nicastrin (NCT), anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2), is assembled in a highly regulated manner and catalyzes the intramembranous proteolysis of many type I membrane proteins, including Notch and amyloid precursor protein. The Notch family of receptors plays important roles in cell fate specification during development and in adult tissues, and aberrant hyperactive Notch signaling causes some forms of cancer. γ-Secretase-mediated processing of Notch at the cell surface results in the generation of the Notch intracellular domain, which associates with several transcriptional coactivators involved in nuclear signaling events. On the other hand, γ-secretase-mediated processing of amyloid precursor protein leads to the production of amyloid β (Aβ) peptides that play an important role in the pathogenesis of Alzheimer disease. We used a phage display approach to identify synthetic antibodies that specifically target NCT and expressed them in the single-chain variable fragment (scFv) format in mammalian cells. We show that expression of a NCT-specific scFv clone, G9, in HEK293 cells decreased the production of the Notch intracellular domain but not the production of amyloid β peptides that occurs in endosomal and recycling compartments. Biochemical studies revealed that scFvG9 impairs the maturation of NCT by associating with immature forms of NCT and, consequently, prevents its association with the other components of the γ-secretase complex, leading to degradation of these molecules. The reduced cell surface levels of mature γ-secretase complexes, in turn, compromise the intramembranous processing of Notch.