Amyloid β protein (Aβ), the major component of neuritic plaques in Alzheimer's disease (AD), is derived from APP by sequential cleavages of β‐ and y‐secretases. Beta‐site APP cleaving enzyme 1 (BACE1) is the major β‐secretase in vivo. Beta‐site APP cleaving enzyme 2 (BACE2) is the homologue of BACE1. The majority of people with Down syndrome (DS)' also called Trisomy 21 syndrome' will develop AD neuropathology after middle age. We and others have shown that APP C99, the major β‐secretase product, and Aβ are markedly increased in DS. Since BACE2 is located on chromosome 21, it is speculated that BACE2 may play a role in AD pathogenesis in DS. In this report we found that BACE2 cleaves APP at a novel θ site downstream of the a site, abolishing Aβ production. Overexpression of BACE2 by lentivirus markedly reduced Aβ production in primary neurons derived from Swedish mutant APP transgenic mice. Despite an extra copy of the BACE2 gene in DS and the increase of its transcription, BACE2 protein levels are unchanged. Our data clearly demonstrate that BACE2, as a novel θ‐secretase to cleave APP within the Aβ domain, is not involved in the AD pathogenesis of DS patients; instead, therapeutic interventions that potentiate BACE2 may prevent AD pathogenesis.—Sun, X., He, G., Song, W. BACE2, as a novel APP θ‐secretase, is not responsible for the pathogenesis of Alzheimer's disease in Down syndrome. FASEB J. 20, 1369–1376 (2006)