Coronary microvascular disease (CMD) is one of the causes of angina, and more postmenopausal women have CMD than premenopausal women. However, the causes of CMD in postmenopausal women are not clear. In this study, we investigate whether and how menopause leads to CMD in female mice. Menopausal mice were generated by administering a 20-day injection of 4-Vinylcyclohexene diepoxide (VCD). The vehicle, sesame oil, was administered to control mice. Coronary flow velocity reserve (CFVR) was measured to assess coronary microvascular function. Experiments were performed at 6 weeks (premenopausal period) and 8 weeks (perimenopausal period) after injection. CFVR was significantly attenuated in VCD-injected mice at 8 weeks after the injection, but not at 6 weeks, compared to the controls. CMD is driven by attenuated vascular relaxation in small CAs and/or decreased capillary rarefaction. We conducted isometric tension in third-order coronary arteries (CAs) and found that endothelium-dependent relaxation (EDR) was significantly attenuated in VCD-injected mice at 8 weeks after injection, but not at 6 weeks, compared to the controls. Capillary density was measured and compared in the left ventricle. Interestingly, there was no significant difference in capillary density between VCD- and vehicle-injected mice at 6 and 8 weeks after injection. Perimenopausal mice, but not premenopausal mice, develop CMD due to attenuated EDR. Regarding impaired EDR, three key genes were identified, Trpv4, Kcna5, and Gja5, by RNA sequencing. This suggests that improving endothelium-dependent relaxation by alternating the key gene expression is the potential mechanism to treat CMD in postmenopausal women.