Alzheimer's disease (AD) is an age-related neurodegenerative disease and the most common form of dementia. AD is pathologically characterized by the deposition of pathogenic Aβ peptides that are derived from larger integral membrane proteins, termed β-amyloid precursor proteins (APPs). In an attempt to understand the function of APP, in vitro studies have focused on the identification of interacting proteins. To investigate the APP in vivo interactome in an unbiased manner, we generated mice that harbor a mouse prion protein promoter-driven cDNA encoding human APP-695 fused to a C-terminal affinity tag. Using this tag, we prepared mild detergent lysates from transgenic mouse brain cortical membrane preparations and isolated a number of previously identified APP-interacting proteins. In addition to these factors, mass spectrometric analysis revealed the presence of NEEP21 as a novel interacting protein. We now report that NEEP21 profoundly affects the processing of APP and Aβ production. Thus, this study demonstrates that using proteomic methods on our transgenic model can uncover important in vivo APP-interacting proteins that will provide insights into the biology of APP.