BACKGROUND: Growing evidence indicates that trimethylamine N -oxide, a gut microbial metabolite of dietary choline and carnitine, promotes both cardiovascular disease and chronic kidney disease risk. It remains unclear how circulating concentrations of trimethylamine N -oxide and its related dietary and gut microbe-derived metabolites (choline, betaine, carnitine, γ-butyrobetaine, and crotonobetaine) affect incident heart failure (HF). METHODS: We evaluated 11 768 participants from the Cardiovascular Health Study and the Multi-Ethnic Study of Atherosclerosis with serial measures of metabolites. Cox proportional hazard models were used to examine the associations between metabolites and incident HF, adjusted for cardiovascular disease risk factors. RESULTS: In all, 2102 cases of HF occurred over a median follow-up of 15.9 years. After adjusting for traditional risk factors, higher concentrations of trimethylamine N -oxide (hazard ratio, 1.15 [95% CI, 1.09–1.20]; P <0.001), choline (hazard ratio, 1.44 [95% CI, 1.26–1.64]; P <0.001), and crotonobetaine (hazard ratio, 1.24 [95% CI, 1.16–1.32]; P <0.001) were associated with increased risk for incident HF. After further adjustment for renal function (potential confounder or mediator), these associations did not reach Bonferroni-corrected statistical significance ( P =0.01, 0.049, and 0.006, respectively). Betaine and carnitine were nominally associated with a higher incidence of HF ( P <0.05). In exploratory analyses, results were similar for subtypes of HF based on left ventricular ejection fraction, and associations appeared generally stronger among Black and Hispanic/Latino versus White adults, although there were no interactions for any metabolites with race. CONCLUSIONS: In this pooled analysis of 2 well-phenotyped, diverse, community-based cohorts, circulating concentrations of gut microbe-derived metabolites such as trimethylamine N -oxide, choline, and crotonobetaine were independently associated with a higher risk of developing HF. REGISTRATION: URL: https://www.clinicaltrials.gov/ ; Unique identifiers: NCT00005133 and NCT00005487.