Background and Purpose: Heart failure with preserved ejection fraction (HFpEF) is a significant global health burden, with limited treatment options. In cardiomyocytes, C-type natriuretic peptide (CNP) stimulation of the guanylyl cyclase B receptor (GC-B) increases cGMP, leading to faster relaxation and decreased diastolic stiffness. We therefore examined whether long-term administration of CNP could prevent diastolic dysfunction in a mouse model of HFpEF. Experimental Approach: C57BL/6N mice were subjected to a combination of a high-fat diet (HFD) and the nitric oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME) in their drinking water to induce HFpEF. Osmotic pumps delivering either CNP or vehicle were implanted for a duration of 6 weeks. Tail-cuff plethysmography, transthoracic echocardiography, pressure volume loop acquisition and morphometrical analysis was used to investigate cardiac function. Key Results: After 6 weeks of CNP treatment, echocardiography and invasive hemodynamic analysis showed that cardiac diastolic function parameters, such as e’, E/e’, end diastolic pressure and end diastolic pressure volume relationship were all markedly ameliorated compared to vehicle-treated HFpEF mice. Vehicle-treated HFpEF mice displayed progressed cardiomyocyte hypertrophy accompanied by increased left ventricular wall thickness and myocardial fibrosis, while those changes were significantly suppressed in CNP-treated mice. Conclusion and Implications: The results demonstrate that CNP treatment prevents cardiac dysfunction and reduces myocardial hypertrophy and fibrosis in HFpEF mice, suggesting its prospective effectiveness for HFpEF treatment.