Abstract Background Vacuolar sorting protein 35 (VPS35), a key component of the retromer, plays an essential role in selectively retrieval of transmembrane proteins from endosomes to trans-Golgi networks. Dysfunctional retromer is a risk factor for neurodegenerative disorders, including Alzheimer’s disease (AD). Microglial VPS35 deficiency is found in AD patients’ brain; however, it remains unclear if and how microglial VPS35-loss contributes to AD development. Methods We used mice with VPS35 cKO (conditional knockout) in microglial cells in 5XFAD, an AD mouse model. The AD related brain pathology (Aβ and glial activation), behavior, and phagocytosis of Aβ were accessed by a combination of immunofluorescence staining analyses and neurological behavior tests. Results A decrease in learning and memory function, but increases in insoluble, fibrillar, and plaques of β-amyloids (Aβ), dystrophic neurites, and reactive astrocytes are observed in microglial VPS35 deficient 5XFAD mice. Further examining microglial phenotype demonstrates necessity of microglial VPS35 in disease-associated microglia (DAM) development and microglial uptake of Aβ, revealing a tight association of microglial Aβ uptake with DAM development. Conclusions Together, these results uncovered a mechanism by which microglial VPS35-deficiency precipitates AD pathology in 5XFAD mice likely by impairing DAM development and DAM mediated Aβ uptake and clearance, and thus accelerating the cognition decline.