Abstract Background Because of its potential influence on the host’s phenotype, increasing attention is paid to organ-specific microbiota in several animal species, including cattle. However, ecosystems other than those related to the digestive tract remain largely understudied. In particular, little is known about the vaginal microbiota of ruminants despite the importance of the reproductive functions of cows in a livestock context, where fertility disorders represent one of the primary reasons for culling. Results In the present study, we aimed at better characterizing the vaginal microbiota of dairy cows through 16S rRNA sequencing, using a large cohort of Holstein cows from Northern France. Our results allowed to define a core microbiota of the dairy cows’ vagina, and highlighted that 90% of the sequences belonged to the Firmicutes, the Proteobacteria, and the Bacteroidetes phyla. The core microbiota was composed of four phyla, 16 families, 14 genera and only one amplicon sequence variant (ASV), supporting the idea of the high diversity of vaginal microbiota within the studied population. This variability was partly explained by various environmental factors such as the herd, the sampling season, the lactation rank and the lactation stage. In addition, we investigated potential associations between the diversity and the composition of the vaginal microbiota and several health-, performance-, and fertility-related phenotypes. Our analyses highlighted significant associations between the α and β- diversities and several traits including the first insemination outcome, the productive longevity, and the culling. Besides, relevant phenotypes were correlated with the abundance of several genera, some of which, such as Leptotrichia , Streptobacillus , Methylobacterium-Methylorubrum , or Negativibacillus , were linked to multiple traits. Conclusion Considering the large number of samples, which were collected in commercial farms, and the diversity of the phenotypes considered, this study represents a first step towards a better understanding of the close relationship between the vaginal and the dairy cow’s phenotypes.