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Introduction Heart failure with preserved ejection fraction (HFpEF) is a multifarious syndrome, accounting for over half of clinical heart failure patients. Neutrophil elastase (NE) has been recently shown to have a detrimental role in multiple vascular diseases. However, little is known about the functional involvement of NE in HFpEF. Herein, we aimed to explore a causal role of NE in HFpEF and unraveling the molecular mechanisms involved.Methods Mice underwent a ‘Two-hit protocol (high-fat diet and Nω-nitro-L-arginine methyl-ester) for 5 and 15 weeks to induce HFpEF. NE-deficiency mice, pharmacologic inhibitor GW311616A, bone marrow transplantation, and adeno-associated virus-9 (AAV9)-mediated in vivo cardiac-specific gene transfer were applied to explore a causal role for NE and associated target gene in HFpEF pathogenesis. Multiple functional and biochemical analyses were conducted to unravel the underlying cellular and molecular mechanisms of NE in HFpEF.Results Mice subjected to ‘Two-hit’ protocol displayed apparent diastolic dysfunctions including increased left ventricular filling pressure, higher lung weight, and poorer exercise performance, but manifested no systolic dysfunctions. ‘Two-hit’ also induced significant increases of neutrophil, macrophages, and inflammatory monocytes in bone marrow, spleen, blood and cardiac tissues. NE cardiac gene expression and protein activity were significantly increased during ‘Two-hit’-induced HFpEF. Data showed that cardiomyocytes could uptake NE produced and secreted from infiltrated inflammatory cells. NE deficiency alleviated ‘Two-hit’-induced diastolic dysfunctions at both 5 and 15 weeks in both male and female mice, and reduced cardiac infiltration of inflammatory cells. Mechanistic data showed that NE mediated cardiac infiltration of inflammatory cells through modulating TBL1x-LTA4H signal axis. Moreover, data from bone marrow transplantation confirmed the major contribution of bone marrow-derived NE to HFpEF. Mechanistically, RNA-binding protein with multiple splicing (RBPMS) has been identified as a novel substrate and functional downstream target of NE in HFpEF. Further mechanistic studies revealed that NE inhibited alternative splicing of Titin mRNA through downregulating RBPMS in the context of HFpEF. Functionally, AAV9-mediated RBPMS cardiac overexpression could significantly alleviates ‘Two-hit’-induced diastolic dysfunctions, recapitulating the functional involvement of NE deficiency in HFpEF. Importantly, AAV9-mediated RBPMS cardiac knockdown could abolish the beneficial effects of NE deficiency on HFpEF phenotype, confirming a critical role of RBPMS in NE-mediated HFpEF phenotypes. Finally, administration of NE pharmacological inhibitor GW311616A significantly ameliorated HFpEF phenotypes.Conclusion We confirm a critical role for NE and its target gene RBPMS in HFpEF. Our findings suggest that NE-RBPMS-Titin signal axis represents a valuable therapeutic for treating patients with HFpEF.