The characterization of vaginal microbiota will help to understand some of the reproductive problems and mechanisms to improve cattle reproduction. The objective of this study was to characterize the vaginal microbiota of cyclical Holstein cows with different parturition orders using 16S rDNA sequencing. Animals were submitted to an estrus synchronization protocol with the use of intravaginal progesterone (P4) implants and were treated or not with ceftiofur hydrochloride. DNA samples were extracted from vaginal swabs on day 0 and 10 of the synchronization, and sequenced with the Illumina MiSeq platform with an average coverage rate of 10.000 reads per samples using a Single-End library for fragments of 300 bp. The main bacterial phyla found in the vaginal tract of Holstein cows, were Firmicutes (37.61%), Tenericutes (29.45%), Proteobacteria (17.47%) and Bacteriodetes (13.73%), followed by Actinobacteria (0.82%) and Spirochaetae (0.45%). The use of intravaginal P4 devices has increased the relative abundance of the genera Family XIII AD3011 and Family XIII unclassified (p < .049). We have also observed an effect of the number of calving on the vaginal microbiota composition, showing that multiparous cows have a greater bacterial diversity than primiparous animals (p < .05). The use of ceftiofur hydrochloride was effective to reduce the vaginal bacteria proliferation. This study describes for the first time the vaginal microbiota of cows synchronized with intravaginal progesterone devices, different from the traditional methods such as microbiological culture and biochemical tests. We have identified a large number of microorganisms commonly found in the gastrointestinal tract of cows, colonizing the vaginal microbiota.