Like humans, canines develop with aging β-amyloid (Aβ) plaques and a progressive cognitive deficit on tasks similar to those used in diagnosis and follow-up of Alzheimer’s disease. Owing to that, dogs are quite unique to investigate the early events taking place in the diffuse Aβ plaque maturation and its relationship with cognitive deficit. The aim of the present investigation was to study the link between the diffuse Aβ plaque maturation and the astro- and microglial reactivity. The involvement of insulin and beta-subunit of S100 protein (S100β) overexpression in the process was also investigated. Aβ plaques were measured and counted in prefrontal cortex of 16 pet dogs of different breeds, weight and sex, classified as control and with a light or severe cognitive deficit. A correlation between canine graded cognitive deficit, diffuse plaque maturation, and S100β (−) astrocytosis, but not with cerebrospinal fluid insulin level, was found that may reflect the very early events of Aβ deposition in Alzheimer’s disease.