AIMS: Trimethylamine N-oxide (TMAO) is a metabolite derived from the gut microbiota. Elevated TMAO levels are associated with a poor prognosis in patients with heart failure with reduced ejection fraction. However, the prognostic effect of elevated TMAO levels on heart failure with preserved ejection fraction (HFpEF) remains unclear. METHODS AND RESULTS: We consecutively enrolled 146 patients who were hospitalized and discharged from Tottori University Hospital with the primary diagnosis of HFpEF (ejection fraction ≥ 50%). High TMAO levels were defined as those greater than the median value in the patients (20.37 μmol/L). Patients with high TMAO levels had a significantly higher prevalence of prior hospitalization for heart failure and severe renal dysfunction than those with low TMAO levels. They also had a significantly higher acylcarnitine to free carnitine ratio than those with low TMAO levels, which indicated abnormal fatty acid metabolism and relative carnitine deficiency. After adjustment for differences in the patients' background in multivariate analysis, high TMAO levels remained independently associated with a high incidence of the composite endpoints of death due to cardiac causes and hospitalization for heart failure (adjusted hazard ratio, 1.91; 95% confidence interval, 1.01 to 3.62; P < 0.05). There was a significant interaction between TMAO and nutritional status on the primary outcome, and the prognostic effect of TMAO was enhanced in patients with malnutrition. CONCLUSIONS: Elevated TMAO levels at discharge are associated with an increased risk of post-discharge cardiac events in patients with HFpEF, especially those with the complication of malnutrition.