Amyloid β peptide (Aβ) generated from amyloid precursor protein (APP) is central to Alzheimer's disease (AD). Signaling pathways affecting APP amyloidogenesis play critical roles in AD pathogenesis and can be exploited for therapeutic intervention. Here, we show that sumoylation, covalent modification of cellular proteins by small ubiquitin-like modifier (SUMO) proteins, regulates Aβ generation. Increased protein sumoylation resulting from overexpression of SUMO-3 dramatically reduces Aβ production. Conversely, reducing endogenous protein sumoylation with dominant-negative SUMO-3 mutants significantly increases Aβ production. We also show that mutant SUMO-3, K11R, which can only be monomerically conjugated to target proteins, has an opposite effect on Aβ generation to that by SUMO-3, which can form polymeric chains on target proteins. In addition, SUMO-3 immunoreactivity is predominantly detected in neurons in brains from AD, Down's syndrome, and nondemented humans. Therefore, polysumoylation reduces whereas monosumoylation or undersumoylation enhances Aβ generation. These findings provide a regulatory mechanism in APP amyloidogenesis and suggest that components in the sumoylation pathway may be critical in AD onset or progression.