γ-Enolase is a neurotrophic-like factor promoting growth, differentiation, survival and regeneration of neurons. Its neurotrophic activity is regulated by cysteine protease cathepsin X which cleaves the C-terminal end of the molecule. We have investigated the expression and colocalization of γ-enolase and cathepsin X in brains of Tg2576 mice overexpressing amyloid precursor protein. In situ hybridization of γ-enolase and cathepsin X revealed that mRNAs for both enzymes were expressed abundantly around amyloid plaques. Immunostaining demonstrated that the C-terminally cleaved form of γ-enolase was present in the immediate plaque vicinity, whereas the intact form, exhibiting neurotrophic activity, was observed in microglia cells in close proximity to senile plaque. The upregulation of γ-enolase in microglial cells in response to amyloid-β peptide (Aβ) was confirmed in mouse microglial cell line EOC 13.31 and primary microglia and medium enriched with γ-enolase proved to be neuroprotective against Aβ toxicity; however, the effect was reversed by cathepsin X proteolytic activity. These results demonstrate an upregulation of γ-enolase in microglia cells surrounding amyloid plaques in Tg2576 transgenic mice and demonstrate its neuroprotective role in amyloid-β-related neurodegeneration.