CD40, a member of tumor necrosis factor receptor superfamily, and its cognate ligand CD40L are both elevated in the brain of Alzheimer's disease (AD) patients compared to controls. We have shown that pharmacological or genetic interruption of CD40/CD40L interaction results in mitigation of AD-like pathology in vivo in transgenic AD mouse models, and in vitro. Recently, we showed that CD40L stimulation could increase Abeta levels via NFkappaB signaling, presumably through TRAFs. In the present work, using CD40 mutants, we show that CD40L can increase levels of Abeta(1-40), Abeta(1-42), sAPPbeta, sAPPalpha and CTFbeta independently of TRAF signaling. We report an increase in mature/immature APP ratio after CD40L treatment of CD40wt and CD40-mutant cells, reflecting alterations in APP trafficking. In addition, results from CD40L treatment of a neuroblastoma cell line over-expressing the C-99 APP fragment suggest that CD40L has an effect on gamma-secretase. Furthermore, inhibition of gamma-secretase activity significantly reduces sAPPbeta levels in the CD40L treated HEK/APPsw CD40wt and the CD40-mutant cells. The latter suggests CD40/CD40L interaction primarily acts on gamma-secretase and affects beta-secretase via a positive feedback mechanism. Taken together, our data suggest that CD40/CD40L interaction modulates APP processing independently of TRAF signaling.