AbstractAccumulating evidence indicates that gut microbiota dysbiosis is associated with increased blood–brain barrier (BBB) permeability and contributes to Alzheimer's disease (AD) pathogenesis. In contrast, the influence of gut microbiota on the blood‐cerebrospinal fluid (CSF) barrier has not yet been studied. Here, we report that mice lacking gut microbiota display increased blood‐CSF barrier permeability associated with disorganized tight junctions (TJs), which can be rescued by recolonization with gut microbiota or supplementation with short‐chain fatty acids (SCFAs). Our data reveal that gut microbiota is important not only for the establishment but also for the maintenance of a tight barrier. Also, we report that the vagus nerve plays an important role in this process and that SCFAs can independently tighten the barrier. Administration of SCFAs in App^NL‐G‐F mice improved the subcellular localization of TJs at the blood‐CSF barrier, reduced the β‐amyloid (Aβ) burden, and affected microglial phenotype. Altogether, our results suggest that modulating the microbiota and administering SCFAs might have therapeutic potential in AD via blood‐CSF barrier tightening and maintaining microglial activity and Aβ clearance.