Although trimethylamine-N-oxide (TMAO) shows a notable correlation with cardiovascular disease, its association with acute ischemic stroke (AIS) remains uncertain and necessitates further investigation.A meta-analysis was conducted to assess the relationship between trimethylamine-N-oxide and acute ischemic stroke.We conducted a comprehensive search in PubMed, Embase, Cochrane, CNKI, VIP, Wanfang, and CBM, spanning from their inception to 23 September 2023. The search was consistently updated and supplemented by bibliographies of retrieved articles and previous reviews. A total of 20 eligible studies, including 17 case‒controls and 3 cohort studies, were selected, involving 9141 participants (5283 case group, 3858 control group). For the dose‒response analysis, three case-control studies were eligible. We extracted and pooled TMAO mean and standard deviation from observational studies for control and ischemic stroke groups. The effect sizes were combined using the random-effects model. Where possible, dose‒response analysis was performed.Overall, the pooled standardized mean difference (SMD) demonstrated significantly higher concentrations of serum/plasma TMAO in AIS compared to the control group (SMD = 1.27; 95% CI: 0.9, 1.61, P<0.001). Additionally, the dose‒response meta-analysis revealed a 12.1% relative increase in the risk of acute ischemic stroke per 1 μmol/L rise in TMAO concentration (RR = 1.12; 95% CI 1.07-1.17; P<0.05; I2 = 1.6%, P = 0.4484).These findings indicate a potential increased risk of AIS associated with elevated TMAO levels.Copyright: © 2023 Hong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.