Fe65 is a brain enriched adaptor protein involved in various cellular processes, including actin cytoskeleton regulation, DNA repair and transcription. A well-studied interacting partner of Fe65 is the transmembrane amyloid-beta precursor protein (APP), which can undergo regulated intramembrane proteolysis (RIP). Following beta and gamma-secretase-mediated RIP, the released APP intracellular domain (AICD) together with Fe65 can translocate to the nucleus and regulate transcription. In this study, we investigated if Fe65 nuclear localization can also be regulated by different alpha-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 differentiated neuroblastoma cells a strong negative impact on Fe65 nuclear localization, equal to the effect observed upon gamma-secretase inhibition, could be detected following inhibition of all three (ADAM9, ADAM10 and ADAM17) alpha-secretases. Moreover, using the comet assay and analysis of Fe65 dependent DNA repair associated posttranslational modifications of histones, we could show that inhibition of alpha-secretase-mediated Fe65 nuclear translocation resulted in impaired capacity of the cells to repair DNA damage. Taken together this suggests that alpha-secretase processing of APP and/or other Fe65 interacting transmembrane proteins play an important role in regulating Fe65 nuclear translocation and DNA repair.