X11 and X11-like proteins (X11L) are neuronal adaptor proteins whose association to the cytoplasmic domain of amyloid β-protein precursor (APP) suppresses the generation of amyloid β-protein (Aβ) implicated in Alzheimer disease pathogenesis. The amyloidogenic, but not amyloidolytic, metabolism of APP was selectively increased in the brain of mutant mice lacking X11L (Sano, Y., Syuzo-Takabatake, A., Nakaya, T., Saito, Y., Tomita, S., Itohara, S., and Suzuki, T. (2006) J. Biol. Chem. 281, 37853-37860). To reveal the actual role of X11 proteins (X11s) in suppressing amyloidogenic cleavage of APP in vivo, we generated X11 and X11L double knock-out mice and analyzed the metabolism of APP. The mutant mice showed enhanced β-site cleavage of APP along with increased accumulation of Aβ in brain and increased colocalization of APP with β-site APP-cleaving enzyme (BACE). In the brains of mice deficient in both X11 and X11L, the apparent relative subcellular distributions of both mature APP and its β-C-terminal fragment were shifted toward the detergent-resistant membrane (DRM) fraction, an organelle in which BACE is active and both X11s are not nearly found. These results indicate that X11s associate primarily with APP molecules that are outside of DRM, that the dissociation of APP-X11/X11L complexes leads to entry of APP into DRM, and that cleavage of uncomplexed APP by BACE within DRM is enhanced by X11s deficiency. Present results lead to an idea that the dysfunction of X11L in the interaction with APP may recruit more APP into DRM and increase the generation of Aβ even if BACE activity did not increase in brain.