Fe65 is a brain enriched adaptor protein involved in various cellular processes, including actincytoskeleton regulation, DNA repair and transcription. A well-studied interacting partner of Fe65is the transmembrane amyloid-β precursor protein (APP), which can undergo regulatedintramembrane proteolysis (RIP). Following β- and γ-secretase-mediated RIP, the released APPintracellular domain (AICD) together with Fe65 can translocate to the nucleus and regulatetranscription. In this study, we investigated if Fe65 nuclear localization can also be regulated bydifferent α-secretases, also known to participate in RIP of APP and other transmembrane proteins.We found that in both Phorbol 12-myristate 13-acetate and all-trans retinoic acid differentiatedneuroblastoma cells a strong negative impact on Fe65 nuclear localization, equal to the effectobserved upon γ-secretase inhibition, could be detected following inhibition of all three (ADAM9,ADAM10 and ADAM17) α-secretases. Moreover, using the comet assay and analysis of Fe65dependent DNA repair associated posttranslational modifications of histones, we could show thatinhibition of α-secretase-mediated Fe65 nuclear translocation resulted in impaired capacity of thecells to repair DNA damage. Taken together this suggest that α-secretase processing of APP and/orother Fe65 interacting transmembrane proteins play an important role in regulating Fe65 nucleartranslocation and DNA repair.