Heart failure with preserved ejection fraction (HFpEF) is a global health epidemic that accounts for half of the heart failure cases. Various therapeutic approaches have been tested to block the activation of the Renin-Angiotensin System (RAS), including AT1R blockers (ARBs), Angiotensin-converting enzyme (ACE) inhibitors (ACEi), and direct renin inhibitors (DRIs) with modest to negligible benefits. The Discovery of ACE2, a novel homolog of ACE, has advanced our understanding of the RAS. ACE2 is a monocarboxypeptidase that degrades Angiotensin II (Ang II) into Angiotensin-(1-7) (Ang-1-7), which works via the activation of the Mas receptor. It has been well understood that the actions of Ang-(1-7) attenuate cardiac remodeling, production of ROS, and cardiac fibrosis. Therefore, the objective of this study was to determine the therapeutic roles of Ang-(1-7) in HFpEF and identify the molecular mechanisms related to its actions. To generate a murine model of HFpEF, 10-12-weeks-old male mice were subjected to a high-fat diet (HFD) (including 60% Kcal fat) in addition to eNOS inhibition with L-NAME (0.5 g/L in drinking water). The Control group received a regular chow diet. The murine model of HFpEF was validated using the non-invasive transthoracic echocardiography and invasive pressure-volume (PV) loop analyses, which exhibited diastolic dysfunction as well as cardiac hypertrophy. To evaluate the effects of Ang-(1-7) on HFpEF, animals were administered with either saline or Ang-(1-7) (24 μg/kg/h) for four weeks. Ang-(1-7) treatment improved diastolic function by reducing isovolumetric relaxation time (IVRT), left ventricular end-diastolic pressure (LVEDP), and Tau value. The development of cardiac hypertrophy in the murine model of HFpEF was confirmed by wheat germ agglutinin (WGA) staining. Furthermore, by investigating different molecular pathways related to the development of hypertrophy, we indicated that the development of hypertrophy, fibrosis, and metabolic dysfunction in this study ii was a result of partial activation of ERK1/2 and inactivation of AMPK, GSK3β, and PDH which did not significantly reverse with 4-weeks treatment of Ang-(1-7).