AIMS: The present study aimed to investigate the direct link between trimethylamine N-oxide (TMAO) and diastolic dysfunction in heart failure with preserved ejection fraction (HFpEF). MATERIALS AND METHODS: Diastolic dysfunction is the main manifestation of HFpEF, so the "two-hit" mouse HFpEF model are used. After treated with high-fat diet (HFD) and N KEY FINDINGS: The HFpEF mice displayed a declined diastolic function, characterized by an increase in the E/E' ratio, accompanied by a significant increase in plasma brain natriuretic peptide levels and cardiac fibrosis and down-regulation of SERCA2 expression, while, DMB treatment improved diastolic function. Subsequently, TMAO was injected intraperitoneally into the mice for 1 month and found that TMAO induced diastolic dysfunction. In addition, we found that either the HFD and L-NAME or TMAO treatment down-regulated Piezo1 expression, and the cardiomyocyte-specific Piezo1 knockout mice (Piezo1 SIGNIFICANCE: In conclusion, our data revealed that TMAO-induced oxidative stress injury by down-regulating Piezo1 to be involve in cardiac diastolic dysfunction of HFpEF. It should be noted that this preclinical study did not evaluate HFpEF-related symptoms such as exercise intolerance or pulmonary congestion, which warrant further validation.