Abstract Body: Background and Purpose: Gut microbiota dysfunction is associated with diabetic cognitive impairment (DCI). However, the mechanisms underlying the interaction of gut microbiota dysbiosis and DCI remain poorly understood. We tested the hypothesis that extracellular vesicles generated by diabetic gut microbiota exacerbate DCI by promoting the impairment of cerebral vascular function. Methods: Gut-EVs from the stools of male non-diabetic dm (dm-gut-EVs) and diabetic (db/db mice) with DCI (db-gut-EVs) mice at 20 weeks of age (20W) were isolated and characterized by means of ultracentrifugation and 16S rRNA sequencing, respectively. Given that db/db mice develop cognitive deficits at 20W, prediabetic db/db mice at 8 weeks of age (8W) were treated with gut-EVs at a dose of 1x10¹⁰ particles/injection intravenously twice a week for 12 weeks. Cognitive performance was assessed using a battery of behavioral tests. Results: 16S rRNA analysis revealed significant alterations in the microbiota composition of db-gut-EVs derived from 20W db/db mice with DCI compared to dm-gut-EVs (n=5/group). Db/db mice treated with db-gut-EVs extracted at 20W, but not with dm-gut-EVs, exhibited a significant decline in learning and memory function, as assayed by the Novel object recognition, Social recognition memory, and Morris water maze assay, starting at 16W and worsening at 20W, compared to db/db mice treated with saline (n=10/group). Additionally, db/db mice treated with db-gut-EVs exhibited increased cerebral vascular thrombosis (18±2 vs 11±2 Fibrin+ vessels/mm² in saline, p<0.05, n=5/group), whereas db/db mice treated with dm-gut-EVs showed a reduction of thrombosis (5±0.5 Fibrin+ vessels/mm², p<0.05). In vitro, treatment of human cerebral endothelial cells (hCECs) with db-gut-EVs significantly (p<0.05) upregulated pro-coagulation and inflammatory proteins, including plasminogen activator inhibitor-1 (PAI-1), tissue factor (TF), NF-κB, and intercellular adhesion molecule 1 (ICAM-1), while reducing tight junction protein ZO-1 expression. Conclusions: Our findings indicate that db-gut-EVs promote DCI by inducing cerebral vascular damage, and underscore a major role for gut microbiota-brain communication in the development of DCI.