Fe65 binds the amyloid precursor protein (APP) and regulates the secretase-mediated processing of APP into several proteolytic fragments, including amyloid β-peptides (Aβ) and APP intracellular domain (AICD). Aβ accumulation in neural plaques is a pathological feature of Alzheimer's disease (AD) and AICD has important roles in the regulation of gene transcription (in complex with Fe65). It is therefore important to understand how Fe65 is regulated and how this contributes to the function and/or processing of APP. Studies have also implicated Fe65 in the cellular DNA damage response with knockout mice showing increased DNA strand breaks and Fe65 demonstrating a gel mobility shift after DNA damage, consistent with protein phosphorylation. In the present study, we identified Fe65 Ser228 as a novel target of the ATM (ataxia telangiectasia mutated) and ATR (ataxia-telangiectasia- and Rad3-related protein) protein kinases, in a reaction that occurred independently of APP. Neither phosphorylation nor mutation of Ser228 affected the Fe65–APP complex, though this was markedly decreased after UV treatment, with a concomitant decrease in the protein levels of APP in cells. Finally, mutation of Ser228 to alanine (thus blocking phosphorylation) caused a significant increase in Fe65–APP transcriptional activity, whereas phosphomimetic mutants (S228D and S228E) showed decreased transcriptional activity. These studies identify a novel phosphorylation site within Fe65 and a novel regulatory mechanism for the transcriptional activity of the Fe65–APP complex.