The incidence of heart failure with preserved ejection fraction (HFpEF) in women significantly increases following menopause. This trend cannot solely be attributed to chronological aging, as evidenced by the more gradual increase in prevalence among men, suggesting that menopause is a provocative event for HFpEF. However, the underlying mechanisms remain elusive and challenging to investigate in human subjects; moreover, an attempt to create HFpEF in ovariectomized (OVX) mice was unsuccessful. In this study, we created an animal model that resembles HFpEF in women undergoing natural menopause. We used 4-vinylcyclohexene dioxide (VCD) to induce "ovary-intact" menopause, combined with the 2hit regimen (HFpEF inducing regimen) to model postmenopausal HFpEF. The female-VCD-2hit mice demonstrate diastolic dysfunction. At the left ventricle (LV) levels, the increased stiffness coefficient of end-diastolic pressure-volume relation (EDPVR), elevated LV end-diastolic pressure, and increased relaxation time constant indicate a heightened LV stiffness, delayed relaxation, and elevated LV filling pressure. At the cardiomyocyte level, the female-VCD-2hit mice exhibit increased cellular diastolic stiffness and delayed relaxation, suggesting that the observed LV dysfunction is derived from the cardiomyocytes. In addition, plasma N-terminal pro-β-type natriuretic peptide (NT-pro-BNP) levels were elevated, whereas Xbp1s transcript levels were reduced, further supporting the existence of HFpEF. Plasma-free testosterone was increased in VCD mice compared with premenopausal and OVX models. Further studies are required to determine whether the relative increase in testosterone is the factor driving HFpEF susceptibility in VCD mice. Ovary-intact postmenopausal status makes female mice vulnerable to HFpEF development. The VCD-2hit model develops a robust HFpEF-like phenotype and is suitable for studying female HFpEF.NEW & NOTEWORTHY Although ovariectomized mice were observed to be resistant to developing HFpEF, ovary-intact postmenopausal mice exhibited an HFpEF-like phenotype under metabolic stress conditions. The increased susceptibility of ovary-intact postmenopausal mice may be due to relative androgen excess conditions, as postmenopausal ovaries retain the ability to secrete androgens. Menopause should be viewed as the imbalance of estrogen and androgens rather than merely an estrogen deficiency, and the role of female androgens in postmenopausal HFpEF warrants further investigation.