Background and Purpose-Trimethylamine N-oxide (TMAO)-a gut derived metabolite-has been shown to be atherogenic. It remains unknown whether TMAO is associated with the risk of first stroke. We aimed to determine the association between serum TMAO levels and first stroke in hypertensive patients without major cardiovascular diseases and examine any possible effect modifiers. Methods-We used a nested case-control design, using data from the CSPPT (China Stroke Primary Prevention Trial), including 622 patients with first stroke and 622 matched controls. The study was conducted from May 2008 to August 2013. The primary outcome was a first stroke. Results-After adjusting for choline, L-carnitine, and other important covariates, including baseline systolic blood pressure and time-averaged systolic blood pressure, during the treatment period, the risk of first stroke increased with each increment of TMAO level (per natural log [TMAO] increment: odds ratio, 1.22; 95% CI, 1.02-1.46). Consistently, compared with participants in the lowest tertile (<1.79 mu mol/L) of serum TMAO levels, a significantly higher risk of first stroke was found in those in higher TMAO tertiles (>= 1.79 mu mol/L; odds ratio, 1.34; 95% CI, 1.00-1.81) or in TMAO tertile 3 (>= 3.19 mu mol/L; odds ratio, 1.43; 95% CI, 1.02-2.01). In the exploratory analysis, we observed an interaction between TMAO and folate levels (>= 7.7 [median] versus <7.7 ng/mL) on first stroke (P for interaction, 0.030). Conclusions-Higher TMAO levels were associated with increased risk of first stroke in hypertensive patients. Our finding, if further confirmed, calls for a carefully designed clinical trial to further evaluate the role of higher TMAO levels on outcomes in hypertensive patients.