To investigate whether elevated plasma trimethylamine N-oxide (TMAO) levels are associated with initial stroke severity and infarct volume. This cross-sectional study included 377 patients with acute ischemic stroke and 50 healthy controls. Plasma TMAO levels were assessed at admission. Stroke infarct size and clinical stroke severity were measured with diffusion-weighted imaging and the NIH Stroke Scale (NIHSS). Mild stroke was defined as an NIHSS score <6. Plasma TMAO levels were higher in patients with ischemic stroke than in healthy controls (median 5.1 vs 3.0 μmol/L; p < 0.001). Every 1-µmol/L increase in TMAO was associated with a 1.13-point increase in NIHSS score (95% confidence interval [CI] 1.04-1.29; p < 0.001) and 1.69-mL increase in infarct volume (95% CI 1.41-2.03; p < 0.001) after adjustment for vascular risk factors. At admission, 159 patients (42.2%) had experienced a mild stroke, and their plasma TMAO levels were lower compared to those with moderate to severe stroke (median 3.6 vs 6.5 µmol/L; p < 0.001). The area under the receiver operating characteristics curve of plasma TMAO level in predicting moderate to severe stroke was 0.794 (95% CI 0.748-0.839; p < 0.001), and the optimal cutoff value was 4.95 μmol/L. The sensitivity and specificity of TMAO levels ≥4.95 μmol/L for moderate to severe stroke were 70.2% and 79.9%, respectively. Patients with ischemic stroke had higher plasma TMAO levels compared to healthy controls. Higher plasma TMAO level at admission is an independent predictor of stroke severity and infarct volume in patients with acute ischemia. © 2020 American Academy of Neurology.