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[w]-nitro-l-arginine methyl ester (L-NAME) for 8 weeks, the cardiac function, myocardial fibrosis, oxidative stress levels, and molecular alterations were assessed. 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 Delta CM) also had diastolic dysfunction. Moreover, the NOX4 expression was up-regulated and the reactive oxygen species levels were increased in the heart tissues of Piezo1 Delta CM or TMAO-treated mice, which was reversed by a Piezo1 activator (Yoda1) in the TMAO-treated mice. Yoda1 also reversed diastolic dysfunction in the HFpEF mice. 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.