Amyloid precursor protein (APP) and its paralogs, amyloid precursor-like protein-1 and amyloid precursor-like protein-2, appear to have redundant but essential role(s) during development. To gain insights into the physiological and possibly pathophysiological functions of APP, we used a functional proteomic approach to identify proteins that interact with the highly conserved C-terminal region of APP family proteins. Previously, we characterized an interaction between APP and ubiquitous mitochondrial creatine kinase. Here, we describe an interaction between APP and a novel protein, 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1). The interaction between APP and NIPSNAP1 was confirmed both in transiently transfected COS7 cells and in the mouse brain, where NIPSNAP1 is expressed at a high level. We demonstrate that NIPSNAP1 is targeted to the mitochondria via its N-terminal targeting sequence, and interacts with mitochondrial chaperone translocase of the outer membrane 22. Mitochondrial localization of NIPSNAP1 appears to be critical for its interaction with APP, and overexpression of APP appeared to disrupt NIPSNAP1 mitochondrial localization. Moreover, APP overexpression resulted in downregulation of NIPSNAP1 levels in cultured cells. Our data suggest that APP may affect mitochondrial function through a direct interaction with NIPSNAP1 as well as with other mitochondrial proteins.