Vaginal atrophy is a prevalent symptom in menopausal women, affecting over 50% of older women and patients with loss of ovarian function. The role of factors other than estrogen, such as the vaginal microbiota (VM), in the development of vaginal atrophy has not been fully explored. Therefore, we selected 8-week-old C57 mice with bilateral ovariectomy for experimentation. After four weeks of treatment, we observed that the vaginal epithelium of ovariectomized mice showed signs of atrophy. There were also significant differences in the structure and metabolites of VM. Vaginal transplantation of microbiota from ovary-intact mice significantly alleviated the vaginal atrophy of ovariectomized mice and altered the structure and metabolism of VM. These findings indicate that ovarian activity significantly affects the structure and metabolism of VM. VM of ovary-intact mice may promote vaginal health by upregulating the estrogen receptor alpha gene (ESR1, one-way ANOVA, F4, 25 = 17.76, P <
0.0001) in vaginal epithelial cells in ovariectomized mice, which in turn promotes cell proliferation (the number of vaginal epithelial cell layers, one-way ANOVA, F4, 25 = 28.04, P <
0.0001). Further studies are needed to investigate the interactions between VM and vaginal health. This finding can help develop new therapeutic strategies and interventions for patients suffering from vaginal atrophy.