Mitochondrial aldehyde dehydrogenase 2 (ALDH2) rs671 variant, in which a G changes to A in the gene, causing a Glu to Lys mutation in the protein, results in a dramatic decrease in the catalytic activity of ALDH2. Population-based data are contradictory about whether ALDH2 rs671 variant increases the risk of Alzheimer’s disease (AD). The prevalence of the ALDH2 rs671 variant is 30%–50% in East Asian populations, but <5% elsewhere. Thus the National Human Brain Bank for Development and Function, the largest brain bank in East Asia, makes it feasible to explore the link between ALDH2 rs671 polymorphism and AD pathology. Here, using 329 human brains, we find that the ALDH2 rs671 variant elevates amyloid beta (Aβ) pathology, with increased plaque deposits and a higher Aβ40/42 ratio, but the overall A-variant is not an independent risk factor for AD. Aggregated Aβ pathology is ALDH2 activity-dependent. As the underlying mechanism, decreased ALDH2 activity leads to increased 4-hydroxy-2-nonenal (4-HNE) accumulation in the brain. The (R)-4-HNE enantiomer alters C99 process by adduction to residue Lys53 of C99 via Schiff-base formation, favoring Aβ40 generation in the Golgi apparatus. Furthermore, decreased ALDH2 activity impaired microglia double edged sword roles in AD brains, both lowered inflammatory factor secretion, as well as Aβ phagocytosis and spreading. We thus clearly define the relationship between ALDH2 rs671 polymorphism and AD, and found ALDH2 rs671 as a key regulator of Aβ40 or Aβ42 generation.