ABSTRACTBacterial vaginosis (BV) is the most common infection of the lower reproductive tract among women of reproductive age, characterized by a depletion of health-associatedLactobacillusand an overgrowth of anaerobes. Metronidazole has been recommended as a first-line therapy for treating BV for decades. Although most cases are cured by the treatment, recurrent infections of BV seriously affect women’s reproductive health. Until now, limited information on the vaginal microbiota has been explored at the species level. Here, we adopted a single molecular sequencing approach for the 16S rRNA gene, named FLAST (full-length assembly sequencing technology), to analyze the human vaginal microbiota that improved species-level resolution for taxonomy and identified microbiota alterations in the vaginal tract in response to treatment with metronidazole. Appling high-throughput sequencing, we identified 96 and 189 novel full-length 16S rRNA gene sequences inLactobacillusandPrevotella, respectively, which had not previously been reported in vaginal samples. Moreover, we found thatLactobacillus inerswas significantly enriched in the cured group before metronidazole treatment, and that was maintained in a high frequency after the treatment, suggesting an important role for this species in response to metronidazole treatment. Our research also highlights the importance of the single-molecule paradigm for progressing the field of microbiology and applying these insights to better understand the dynamic microbiota during BV treatment. Subsequent novel treatment approaches should be proposed to improve BV treatment outcomes, optimize the vaginal microbiome, and reduce gynecological and obstetric sequelae.IMPORTANCEBacterial vaginosis (BV) is a common infectious disease of the reproductive tract. Metronidazole treatment, as the first line of treatment, frequently fails at recovery of the microbiome. However, the precise types ofLactobacillusand other bacteria involved in BV remain unclear, and this has resulted in a failure to identify potential markers to predict clinic outcomes. In this study, we adopted a 16S rRNA gene full-length assembly sequencing technology for the taxonomy analysis and evaluation of vaginal microbiota before and after treatment with metronidazole. We additionally identified 96 and 189 novel 16S rRNA gene sequences inLactobacillusandPrevotellaspecies, respectively, in vaginal samples, which improves our understanding of the vaginal microbiota. Moreover, we found that the abundance ofLactobacillus inersandPrevotella biviabefore treatment was associated with a lack of cure. These potential biomarkers will help to facilitate future studies aimed at improving BV treatment outcomes, optimize the vaginal microbiome, and reduce adverse sexual and reproductive outcomes.