Alzheimer’s disease is a neurodegenerative disease characterized by aberrant proteolysisof the transmembrane amyloid-β precursor protein (APP). The brain enriched adaptor protein Fe65interacts with APP and participates together with APP and/or APP fragments in a number ofcytoplasmic and nuclear functions. However, how the Fe65 subcellular localization, interactionwith APP/APP fragments are regulated, as well as how Fe65 influences APP processing, is stillnot fully understood. In this study, we investigated the effect of serine 228 phosphorylation onFe65 nuclear localization, APP interaction and APP processing. We show that although a serine228 phosphomimicking variant of Fe65 (Fe65-S2285E) was not excluded from the nucleus, a trendtowards reduced nuclear level and nuclear/cytoplasmic ratio of this mutant could be observed,suggesting that phosphorylation of serine 288 could participate in regulation of the Fe65subcellular localization. Interestingly, we found that not only Fe65-S2285E, but also mutation ofserine 228 to alanine (Fe65-S228A) resulted in a similar and dramatic increase of the Fe65interaction with full-length APP. Moreover, we found that this increased APP interaction resultedin reduced α-secretase processing of APP and thus less generation of the neuroprotective sAPPαfragment. This suggest that the N-terminal domain of Fe65 may have a more prominent role inmediating the Fe65-APP interaction and regulating APP processing than previously thought.