Fe65 is an adaptor protein that binds to the amyloid precursor protein (APP) within the 82YENPTY687 motif of APP which is important for amyloid β (Aβ) production. Considering that Fe65 binds to this motif, it can be hypothesized that Fe65 may influence the trafficking of APP and hence its processing by α- and/or β-secretase. Therefore in this study we wanted to determine how knock-down of Fe65 effects the processing of endogenous APP in human SH-SY5Y neuroblastoma cells. Our results showed that Fe65 knock-down did not have any effect on sAPPα secretion in SH-SY5Y cells. However, decreased levels of membrane-bound APP stubs C83 and C99 were observed, suggesting that Fe65 has a stabilizing effect on the C-terminal fragments. Furthermore, we wanted to investigate effects of retinoic acid (RA)-induced neronal differentiation on Fe65 expression. It has previously been shown that under these conditions mRNA and protein levels of APP increase concomitant with increased secretion of sAPPα, shifting the processing of APP to the more non-amyloidogenic pathway. We observed that RA-induced neuronal differentiation increases the protein levels of Fe65 in SH-SY5Y cells and gives rise to an electrophoretic mobility shift due to increased phosphorylation. The increased expression levels of Fe65 during neuronal differentiation concomitant with the increase of Fe65 phosphorylation, suggest that Fe65 and its phosphorylation may play a role during neuronal differentiation.