Background: Premature ovarian insufficiency (POI) is a complex reproductive endocrine disease that can affect multiple systems. It is highly heterogeneous in etiology and the exact etiology remains unclear. Infectious factors may be related to POI, but researches on the alterations of microbiome in POI patients are scarce. Results: Vaginal swabs were collected from 52 POI patients (29 for sequencing, 23 for validation) and 46 healthy individuals of comparable age (26 for sequencing, 20 for validation). 16S rRNA gene sequencing targeting the V3-V4 hypervariable regions was performed to evaluate the alterations of vaginal microbiota in POI patients. The relative abundance of Actinobacteria (23.34% vs 10.65%, P=0.017), Atopobium (11.11% vs 0.01%, P﹤0.001), and Gardnerella (8.05% vs 3.14%, P=0.002) were significantly increased in POI patients, while Bifidobacterium (3.95% vs 7.44%, P=0.017) was significantly decreased. Cluster analysis of dominant strains showed that the proportion of POI patients whose predominant bacteria were not Lactobacillus was increased than the control group, but the difference was not statistically significant (8/29 vs 2/26, P=0.105). More interestingly, these changes in vaginal microbiota were significantly correlated with declined ovarian function in POI patients, including decreased ovarian reserve, ovarian endocrine disruption, and symptoms of perimenopausal syndrome. Actinobacteria, Atopobium, and Gardnerella appeared to be detrimental to ovarian function, while Bifidobacterium seemed to be benificial. Conclusions: The present study revealed the correlation between vaginal microbiota and POI, fill the gap in the field of microbial and POI research and provide a new research strategy for POI.