Post-menopausal women exhibit an exponential increase in the incidence of heart failure with preserved ejection fraction compared with men of the same age, which indicates a potential role of hormonal changes in subclinical and clinical diastolic dysfunction. This paper reviews the preclinical evidence that demonstrates the involvement of estrogen in many regulatory molecular pathways of cardiac diastolic function and the clinical data that investigates the effect of estrogen on diastolic function in post-menopausal women. Published reports show that estrogen deficiency influences both early diastolic relaxation via calcium homeostasis and the late diastolic compliance associated with cardiac hypertrophy and fibrosis. Because of the high risk of diastolic dysfunction and heart failure with preserved ejection fraction in post-menopausal women and the positive effects of estrogen on preserving cardiac function, further clinical studies are needed to clarify the role of endogenous estrogen or hormone replacement in mitigating the onset and progression of heart failure with preserved ejection fraction in women.