AbstractAlzheimer's disease (AD), the most prevalent neurodegenerative disorder worldwide, is clinically characterized by cognitive deficits. Neuropathologically, AD brains accumulate deposits of amyloid‐β (Aβ) and tau proteins. Furthermore, these misfolded proteins can propagate from cell to cell in a prion‐like manner and induce native proteins to become pathological. The entorhinal cortex (EC) is among the earliest areas affected by tau accumulation along with volume reduction and neurodegeneration. Neuron–glia interactions have recently come into focus; however, the role of microglia and astroglia in the pathogenesis of AD remains unclear. Proteomic approaches allow the determination of changes in the proteome to better understand the pathology underlying AD. Bioinformatic analysis of proteomic data was performed to compare ECs from AD and non‐AD human brain tissue. To validate the proteomic results, western blot, immunofluorescence, and confocal studies were carried out. The findings revealed that the most disturbed signaling pathway was synaptogenesis. Because of their involvement in synapse function, relationship with Aβ and tau proteins and interactions in the pathway analysis, three proteins were selected for in‐depth study: HSP90AA1, PTK2B, and ANXA2. All these proteins showed colocalization with neurons and/or astroglia and microglia and with pathological Aβ and tau proteins. In particular, ANXA2, which is overexpressed in AD, colocalized with amoeboid microglial cells and Aβ plaques surrounded by astrocytes. Taken together, the evidence suggests that unbalanced expression of HSP90AA1, PTK2B, and ANXA2 may play a significant role in synaptic homeostasis and Aβ pathology through microglial and astroglial cells in the human EC in AD.