Objectives To investigate whether the plasma level of trimethylamine N-oxide (TMAO), a proatherosclerotic intestinal microbiota metabolite, can be a predictor of ischemic brain injury secondary to carotid artery stenting (CAS). Methods In this multicenter, prospective cohort study, we enrolled patients with severe carotid artery stenosis (>70%) who were prepared for CAS. Plasma TMAO level was measured within 3 days before CAS, and MRI was performed 1 to 3 days after CAS. Results The mean age of the 268 eligible patients was 64.4 years. New lesions on diffusion-weighted imaging (DWI) were detected in 117 patients (43.7%). TMAO level was higher in patients with new (DWI) lesions than in patients without new lesions (median 5.2 vs 3.2 mu mol/L; p < 0.001). Increased plasma TMAO levels were associated with an increased risk of having new lesions on DWI after CAS (adjusted odds ratio for the highest vs lowest quartile, 3.85; 95% confidence interval, 1.37-7.56, p < 0.001; adjusted odds ratio for the third vs lowest quartile, 1.86; 95% confidence interval, 1.09-4.66, p = 0.02). The area under the receiver operating characteristic curve of TMAO was 0.706 for new lesions on DWI, and the optimal cutoff value was 4.29 mu mol/L. The sensitivity, specificity, positive predictive value, and negative predictive value of TMAO levels = 4.29 mu mol/L for predicting new lesions on DWI were 61.5%, 74.8%, 65.5%, and 65.5%, respectively. Conclusions Increased TMAO levels are associated with an increased risk of new ischemic brain lesions on post-CAS MRI scans.