BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) has emerged as one of the most challenging public health issues worldwide, with few effective preventive and therapeutic options available. S100A8/A9 is an inflammatory mediator that contributes to the development of several cardiovascular diseases via the TLR4/NF-κB pathway. This study aimed to investigate the role of S100A8/A9 in HFpEF and unravel potential mechanisms. METHODS: The HFpEF model was established in C57BL6/J mice by administering a high-fat diet combined with NG-nitroarginine methyl ester hydrochloride (L-NAME). From the eighth week, mice were treated with the S100A8/A9 inhibitor paquinimod (ABR 215757) via oral gavage for 8 weeks. Body weight, tibial length, glucose tolerance, and blood lipids were measured following the intervention. Subsequently, cardiac function, myocardial hypertrophy, cardiac fibrosis, inflammatory markers, and oxidative stress were evaluated. AAV9-TLR4-shRNA and the TLR4 inhibitor TAK-242 were employed in vivo and in vitro, respectively, to explore the probable underlying mechanism. RESULTS: The results showed that paquinimod improved diastolic dysfunction in HFpEF mice without affecting systolic function. It also mitigated cardiomyocyte hypertrophy, suppressed cardiac fibrosis, decreased myocardial inflammation, and attenuated oxidative stress. Mechanistically, paquinimod inhibited the TLR4/NF-κB signalling pathway, and TLR4 inhibition reversed the effects of S100A8/A9 in vitro and partly alleviated the heart failure phenotype in mice with HFpEF. CONCLUSIONS: Inhibition of S100A8/A9-mediated inflammation improved diastolic function and reversed the pathological changes of the heart in mice with HFpEF. The observed effects were potentially mediated via inhibition of the TLR4/NF-κB pathway. The present study identified S100A8/A9 as a possible therapeutic target in HFpEF.