Introduction Communication between the gut microbiota and the brain is primarily mediated via soluble microbe-derived metabolites, but the details of this pathway remain poorly defined. Methylamines produced by microbial metabolism of dietary choline and L-carnitine have received attention due to their proposed association with vascular disease, but their effects upon the cerebrovascular circulation have not hitherto been studied. Aim Here we use an integrated in vitro/in vivo approach to investigate how physiologically relevant concentrations of the dietary methylamine trimethylamine (TMA) and its host-derived oxidation product trimethylamine N-oxide (TMAO) affect blood-brain barrier (BBB) integrity. Methods hCMEC/D3 human immortalised cerebromicrovascular endothelial cells grown under polarising conditions on 0.4μm transwell filters were used to model BBB barrier function in vitro, assessing permeability to a 70kDa FITC-dextran tracer, transendothelial electrical resistance (TEER), and gene expression by microarray, n=3-4 independent experiments. Male wild-type C57Bl/6 mice were used to assess in vivo effects of acute and chronic TMAO exposure and interactions with lipopolysaccharide (LPS)-induced inflammatory BBB disruption, monitoring BBB integrity via Evans blue dye extravasation and brain gene expression by RNAseq, n=5-8 mice. All experiments were conducted under UK Home Office licence approval and reviewed by the QMUL Animal Welfare Ethical Review Board. Statistical analysis was by one- or two-way ANOVA as appropriate, with P<0.05 taken as statistically significant. Gene expression data was corrected for multiple testing using the Benjamini-Hochberg procedure and P<0.1 was taken as statistically significant. Results In vitro studies revealed a clear distinction between the effects of TMA and TMAO; TMA dose-dependently impaired BBB permeability barrier function, whilst TMAO exhibited a biphasic response, physiologically relevant concentrations reducing and supra-physiological doses enhancing permeability. Microarray analysis indicated that TMA activated pathways characteristic of cellular stress responses, while TMAO upregulated a number of pathways associated with cytoskeletal rearrangement and actin bundle formation. Notably, TMAO upregulated expression of the major tight junction regulator annexin A1. Further analysis of this pathway using hCMEC/D3 cells stably expressing shRNA sequences targeting annexin A1 showed this protein to be a major mediator of TMAO actions. Acute treatment of mice with TMAO (1.8mg/kg body weight, i.p.) enhanced BBB integrity within 2 hours, and was able to restore the BBB permeability defect induced by LPS administration (3mg/kg i.p., assessed 4h post-LPS, TMAO administered 2h post-LPS). Whole brain RNAseq analysis of TMAO-treated animals identified upregulation of multiple genes involved in cellular or axonal growth pathways. Chronic treatment of mice for two months with TMAO via the drinking water (0.5mg/l) reduced signs of BBB integrity damage caused by long-term sub-acute LPS treatment (0.5 mg/kg/week, i.p.). Conclusions We show that physiologically-relevant concentrations of the dietary methylamine TMAO can beneficially modulate BBB integrity, providing direct mechanistic evidence for a positive role of this microbiome-associated metabolite and emphasising the BBB as an interface in the gut-brain axis. Notably, our findings stand in contrast to previous work describing deleterious effects of TMAO exposure at high concentrations or under non-physiological conditions, emphasising the importance of taking a holistic approach to understanding gut microbiota-host interactions.