Alzheimer's disease is increased in diabetic patients. A defective insulin activity on the brain has been hypothesized to contribute to the neuronal cell dysregulation leading to AD, but the mechanism is not clear. We analyzed the effect of insulin on several molecular steps of amyloid precursor protein (APP) processing and β-amyloid (Aβ) intracellular accumulation in a panel of human neuronal cells and in human embryonic kidney 293 cells overexpressing APP-695. The data indicate that insulin, via its own receptor and the phosphatidylinositol-3-kinase/AKT pathway, influences APP phosphorylation at different sites. This rapid-onset, dose-dependent effect lasts many hours and mainly concerns dephosphorylation at the APP-T668 site. This effect of insulin was confirmed also in a human cortical neuronal cell line and in rat primary neurons. Cell fractionation and immunofluorescence studies indicated that insulin-induced APP-T668 dephosphorylation prevents the translocation of the APP intracellular domain fragment into the nucleus. As a consequence, insulin increases the transcription of antiamyloidogenic proteins such as the insulin-degrading enzyme, involved in Aβ degradation, and α-secretase. In contrast, the transcripts of pro-amyloidogenic proteins such as APP, β-secretase, and glycogen synthase kinase (Gsk)-3β are decreased. Moreover, cell exposure to insulin favors the nonamyloidogenic, α-secretase-dependent APP-processing pathway and reduces Aβ40 and Aβ42 intracellular accumulation, promoting their release in the extracellular compartment. The latter effects of insulin are independent of both Gsk-3β phosphorylation and APP-T668 dephosphorylation, as indicated by experiments with Gsk-3β inhibitors and with cells transfected with the nonphosphorylatable mutated APP-T668A analog. In human neuronal cells, therefore, insulin may prevent Aβ formation and accumulation by multiple mechanisms, both Gsk-3β dependent and independent.