Accumulation of insoluble deposits of amyloid β-peptide (Aβ), derived from amyloid precursor protein (APP) processing, represents one of the major pathological hallmarks of Alzheimer's disease (AD). Perturbations in APP transport and hydrolysis could lead to increased Aβ production. However, the precise mechanisms underlying APP transport remain elusive. The GDP dissociation inhibitor2 (GDI2), a crucial regulator of Rab GTPase activity and intracellular vesicle and membrane trafficking, was investigated for its impact on AD pathogenesis through neuron-specific knockout of GDI2 in 5xFAD mice. Notably, deficiency of GDI2 significantly ameliorated cognitive impairment, prevented neuronal loss in the subiculum and cortical layer V, reduced senile plaques as well as astrocyte activation in 5xFAD mice. Conversely, increased activated microglia and phagocytosis were observed in GDI2 ko mice. Further investigation revealed that GDI2 knockout led to more APP co-localized with the ER rather than the Golgi apparatus and endosomes in SH-SY5Y cells, resulting in decreased Aβ production. Collectively, these findings suggest that GDI2 may regulate Aβ production by modulating APP intracellular transport and localization dynamics. In summary, our study identifies GDI2 as a pivotal regulator governing APP transport and process implicated in AD pathology; thus highlighting its potential as an attractive pharmacological target for future drug development against AD.