Abstract Body (Do not enter title and authors here): Background: Recently we described the first retro-enantiomer (RE) of the renin angiotensin system (RAS), a mimetic peptide of angiotensin-(1-9) [Ang-(1-9)RE]. It binds to the AT2 receptor and exhibits increased chemical stability in human serum with in vivo antihypertensive, cardioprotective, and antihypertrophic properties compared to Ang-(1-9) (Circulation 2024 3;150(10):816-820). However, the effects of Ang-(1–9)RE on blood pressure, cardiac structural abnormalities and dysfunction, exercise tolerance and myocardial infiltration by activated macrophages in heart failure with preserved ejection fraction (HFpEF) are unknown. Aim. To determine whether Ang-(1-9)(RE) administration prevents hypertension and cardiac damage, as well as diastolic dysfunction and pro-inflammatory macrophages (CCR2⁺MHCII⁺) infiltration in preclinical HFpEF. Methods: 12-week-old male C57BL/6N mice were randomized to 3 experimental groups: control diet, high-fat diet (HFD, 60% kcal from fat)+ L-NAME (L,0.65 g/L in drinking water) and HFD+L+Ang-(1-9)RE [1200 ng/kg min] delivered by osmotic minipumps for 15 weeks. Body weight (BW), systolic (SBP) and diastolic blood pressure (DBP), glucose tolerance (GT), exercise tolerance (ET), cardiac function and cardiac pro-inflammatory macrophages CCR2⁺MHCII⁺ were determined. Results: (mean ± SEM, n=8-12/group). Compared with HFD+L, HFD+L+Ang-(1-9)RE mice showed less SBP (137±4 vs 155±3, p<0.01), DBP (96±4 vs 128±2, p<0.01) and area under the curve of the GT (p <0.001, F=22.4). HFD+L+Ang-(1-9)RE vs HFpEF mice walked a longer distance on the treadmill (473±31 vs 365±28 mts, p<0.01). Both septal and posterior wall thickness were greater in the HFpEF vs HFD+L+Ang-(1-9)RE mice (p<0.0001, F=10.6 and p<0.0001, F=14.6, respectively). In HFD+L+Ang-(-9)RE vs HFpEF mice left atrium size and diastolic dysfunction decreased (p<0.001, F=35.6). Ejection fraction was normal in all groups. In the myocardium of HFD+L+Ang-(-9)RE mice, CCR2⁺MHCII⁺ macrophages were significantly decreased compared to HFpEF mice (p<0.001, F=12.7). Conclusion: Ang-(1-9)RE peptide, the first RE for the RAS prevented the HFpEF by reducing blood pressure and glucose intolerance, diastolic dysfunction, cardiac hypertrophy and the abundance of pro-inflammatory macrophages in the myocardium. These results open an avenue for the design of new families of mimetic peptides that could contribute to improve HFpEF therapy. FONDECYT 1221585, FONDECYT 1231604, ANILLO ACT240058, FONDAP 1523A0008.