Processing of APP is a well-acknowledged central pathogenic mechanism in Alzheimer’s disease. However, influences of age-associated cellular alterations on the biochemistry of APP processing are not studied in molecular detail so far. Here, we report that processing of endogenous APP is down-regulated during aging of normal human fibroblasts (IMR-90). The generation of intracellular APP cleavage products, C99, C83 and AICD, was gradually declining with increasing lifespan and was accompanied by a reduced secretion of sAPP and sAPPa. Further, the maturation of APP was reduced in senescent cells, which has been shown to be directly mediated by age-associated increased cellular cholesterol levels. Of the APP processing secretases protein levels of constituents of the -secretase complex, PS1 and nicastrin, were progressively reduced during aging, resulting in a progressive decrease in -secretase enzymatic activity. ADAM10 and BACE protein levels exhibited no age-associated regulation but, interestingly, BACE enzymatic activity was even increased in aged cells. PS1 and BACE are located in detergent resistant membranes (DRMs), well-structured membrane microdomains exhibiting high levels of cholesterol and caveolin-1. Although total levels of both structural components of DRMs were up-regulated in aged cells, their particular DRM association was decreased. This age-dependent membrane modification was associated with an altered distribution of PS1 and BACE between DRM and non-DRM fractions, very likely affecting their APP processing potential. In conclusion, we have found a significant modulation of endogenous APP processing and maturation in human fibroblasts caused by age-associated alterations in cellular biochemistry.