: Accumulating evidence suggests that Trimethylamine-N-oxide (TMAO), a gut microbial metabolite, is implicated in the pathogenesis of many cardiovascular diseases. The aim of the present study was to investigate the serum levels of TMAO in Chinese patients with ischemic stroke.: In the present study, 255 consecutive patients with first-ever acute ischemic stroke and 255 age and gender-matched healthy volunteers were included for testing serum TMAO. Stroke severity was determined by the NIH Stroke Scale (NIHSS). The stroke severity was dichotomized as minor (NIHSS ≤ 5) and moderate-to-high clinical severity (NIHSS > 6).: The serum levels of TMAO in stroke ranged from 0.5 to 18.3 μM, with a median value of 5.8 (interquartile range (IQR), 3.3-10.0) μM, which was higher than in those controls (3.9; IQR, 2.6-6.1 μM). The median level of TMAO in those patients was significantly lower than in those moderate-to-high stroke patients (4.1 μM [IQR, 2.8-6.2] vs. 9.1 μM [5.1-11.0]; <0.001). In univariate and multivariable models, the unadjusted risk of moderate-to-high stroke was increased by 31% (odds ratio (OR) = 1.31 [95% confidence interval (CI): 1.21-1.42], <0.001) and 22% (OR = 1.22; 95% CI = 1.08-1.32; <0.001), when TMAO was increased each by 1 μM. Based on the receiver operating characteristic (ROC) curve, the optimal cut-off value of serum level of TMAO as an indicator for screening of moderate-to-high stroke was estimated to be 6.6 μM, which yielded a sensitivity of 69.3 % and a specificity of 79.0%, with the area under the curve at 0.750 (95% CI, 0.687-0.812).: Higher TMAO levels were associated with increased risk of first ischemic stroke and worse neurological deficit in Chinese patients.