Abstract Body: Heart failure with preserved ejection fraction (HFpEF) can develop with obesity, hyperinsulinemia, and glucose intolerance or type 2 diabetes, a phenogroup designated cardiometabolic HFpEF. While inflammation considered an important underlying mechanism, the role of innate immunity is poorly define. We investigated the role of macrophages in pathogenesis of cardiometabolic HFpEF. C57BL/6 mice were fed either high-fat diet (HFD, 45% kcal fat, 35% carbohydrate, 20% protein, 4.73 kcal/g) or control diet (CD, 10% kcal fat, 70% carbohydrate, 20% protein, 3.85 kcal/g) for 24-28 weeks. HFD mice exhibited significantly increased body weight (BW) at equivalent calorie consumption, increased white adipose tissue (WAT) mass, glucose intolerance, and hyperinsulinemia. HFD mice also exhibited HFpEF, evidenced by increased relative wall thickness (RWT), E/e’ ratio, LA area, LVEDP, and lung weight and decreased exercise tolerance with preserved EF. CD64⁺MHCII⁺LyVE1⁺CCR2^– resident and CD64⁺MHCII⁺CCR2⁺ infiltrating macrophages expanded significantly in both cardiac and visceral fat tissue. To evaluate the role of macrophages in HFpEF, we used Macrophage Ablation Fas-Induced Apoptosis (MaFia) mice that allow for conditional depletion of mononuclear phagocytes with the Fas ligand dimerizer AP20187. AP20187 treatment after 24 weeks of HFD feeding in MaFia mice depleted macrophages and normalized BW, and reversed cardiac hypertrophy (decreased RWT, histological myocyte cross-sectional area [CSA]), and cardiac dysfunction (improved E/e’ ratio and global longitudinal strain [GLS]), as compared to vehicle-treated HFD-induced HFpEF MaFia mice. To establish sufficiency of cardiac macrophages, we FACS-sorted CD45⁺CD64⁺F4/80⁺ cardiac macrophages from HFD and CD mice, and adoptively transferred (10K cells/mouse) by intramyocardial injection into naïve recipient mice, with evaluation 4 w later. Mice with HFD-heart cardiac macrophage transfer evidenced significant increases in left atrial area (P=0.008, E/e’ ratio (P=0.016), cardiomyocyte CSA (P=0.015) and cardiac fibrosis (P<0.05), and reduced |GLS|, as compared to recipient mice with CD-heart cardiac macrophage transfer. There were no differences in body weight between the two groups. We conclude: cardiac macrophage expansion is central to the pathogenesis of cardiometabolic HFpEF, and are both necessary and sufficient for the cardiac phenotype. These results raise the potential for macrophage-targeted interventions to mitigate HFpEF.