Trimethylamine N-oxide (TMAO) has emerged as a newly identified gut microbiota-dependent metabolite contributing to a variety of diseases, such as diabetes, atherosclerosis, and cardiovascular diseases. The aim of our study was to determine whether a relatively high TMAO level is associated with an increased risk of poor outcome in ischemic stroke patients. From June 2018 to December 2018, we prospectively recruited acute ischemic stroke patients diagnosed within 24 h of symptom onset. The plasma TMAO level was measured at admission for all patients. Functional outcome was evaluated at 3 months after the stroke using the modified Rankin Scale (mRS) and then dichotomized as favorable (mRS 0-2) or unfavorable (mRS 3-6). A multivariate logistic regression analysis was conducted to evaluate the association between TMAO concentration and poor functional outcome and mortality at 3 months. Of the 225 acute ischemic stroke patients included in the analysis, the median TMAO concentration was 3.8 µM (interquartile range, 1.9-4.8 µM). At 3 months after admission, poor functional outcome was observed in 116 patients (51.6%), and 51 patients had died (22.7%). After adjusting for potential confounders, patients with TMAO levels in the highest quartile were more likely to have higher risks of poor functional outcome [compared with the lowest quartile, odds ratio (OR) 3.63; 95% confidence interval (CI) 1.34-9.82; P = 0.011] and mortality (OR 4.27; 95% CI 1.07-17.07; P = 0.040). Our data suggest that a high plasma TMAO level upon admission may predict unfavorable clinical outcomes in acute ischemic stroke patients.