AbstractAlzheimer's disease is the most frequent neurodegenerative disorder in the aged population and is characterized by the deposition of the 40/42‐residue amyloid β protein (Aβ), a proteolytic fragment of the β‐amyloid precursor protein (APP). A common apolipoprotein E (apoE) polymorphism is associated with an increased risk of developing the disease. In order to assess the putative relationship between apoE and amyloidogenesis in the CNS, we prepared primary cortical neurons overexpressing humanized APP695 bearing the Swedish mutation (hAPP695sw) and we analysed APP expression and processing after: (i) coculture with primary astrocytes from wild‐type, apoE‐deficient (E0) mice, or mice overexpressing human apoE2, E3, or E4; (ii) treatment with conditioned media from apoE0, E2, E3 or E4 astrocytes; and (iii) treatment with human recombinant ApoE or human apoE purified from conditioned media of stably transfected RAW264 cells (E2, E3 and E4). Interestingly, a strong decrease in APP expression was observed only when neurons were cocultured with astrocytes (and independently of the apoE genotype considered), suggesting that cell–cell contact is required. Moreover, apoE4‐secreting astrocytes, but not recombinant or purified apoE4, significantly increased Aβ production and decrease sAPPα secretion only when cultured in direct contact with neurons, whereas apoE2 astrocytes had a protective effect. We conclude that astrocytes: (i) strongly regulate neuronal APP expression in primary neurons, and (ii) promote the amyloidogenic pathway in an apoE4‐dependent manner. Thus, apoE and astrocytic factor(s) may modulate the pathogenesis of Alzheimer's disease.