The phosphotyrosine binding domain of the neuronal protein X11α/mint-1 binds to the C-terminus of amyloid precursor protein (APP) and inhibits catabolism to β-amyloid (Aβ), but the mechanism of this effect is unclear. Coexpression of X11α or its PTB domain with APPswe inhibited secretion of Aβ40 but not APPsβswe, suggesting inhibition of γ- but not β-secretase. To further probe cleavage(s) inhibited by X11α, we coexpressed γ-secretase (BACE-1) or a component of the γ-secretase complex (PS-1Δ9) with APP, APPswe, or C99, with and without X11α, in HEK293 cells. X11α suppressed the PS-1Δ9-induced increase in Aβ42 secretion generated from APPswe or C99. However, X11α did not impair BACE-1-mediated proteolysis of APP or APPswe to C99. In contrast to impaired γ-cleavage of APPswe, X11α or its PTB domain did not inhibit γ-cleavage of NotchΔE to NICD (the Notch intracellular domain). The X11α PDZ-PS.1Δ9 interaction did not affect γ-cleavage activity. In a cell-free system, X11α did not inhibit the catabolism of APP C-terminal fragments. These data suggest that X11α may inhibit Aβ secretion from APP by impairing its trafficking to sites of active γ-secretase complexes. By specifically targeting substrate instead of enzyme X11α may function as a relatively specific γ-secretase inhibitor.