A multi-kingdom collection of 33,804 reference genomes for the human vaginal microbiome has been established, providing a comprehensive resource for studying the vaginal microbiome and its impact on health. The Vaginal Microbial Genome Collection (VMGC) includes 19,542 prokaryotic genomes, 38 fungal genomes, and 14,224 viral genomes, spanning 786 prokaryotic species, 11 fungal species, and 4,263 viral operational taxonomic units (vOTUs). Over 25% of prokaryotic species and 85% of viral vOTUs remain uncultured, highlighting the need for further research. The VMGC significantly enriches genomic diversity, particularly for prevalent vaginal pathogens such as BVAB1 and Amygdalobacter spp. It offers insights into the functional traits of prokaryotes, including the underexplored Saccharofermentales order, and eukaryotic viruses, revealing their niche adaptation and potential roles in the vagina. The VMGC serves as a valuable resource for studying vaginal microbiota and its impact on vaginal health. The human vagina is a diverse ecosystem hosting bacteria, viruses, fungi, and other micro-eukaryotes crucial for maintaining women's and fetal health. In healthy women, Lactobacillus species dominate the vaginal microbiota, producing lactic acid to maintain an acidic environment and prevent harmful microorganisms. Dysbiosis often leads to bacterial vaginosis, characterized by the overgrowth of anaerobic bacteria. Early research relied on culture-based or real-time PCR methods, which had limitations in capturing microbial diversity. Next-generation sequencing has advanced understanding, uncovering uncultivated microorganisms associated with vaginal health. Whole metagenomic sequencing has elucidated the compositional and functional diversity of the vaginal microbiome. The Human Microbiome Project has provided an overview of microbial and functional profiles across various body sites, including the vagina. The study by Jie et al. has uncovered associations between the vaginal microbiota and life history, while Fettweis et al. linked the vaginal microbiota to preterm birth. The VMGC was constructed by integrating available metagenomic datasets and microbial genomes, along with in-house cultivation of vaginal fungi. The VMGC comprises 19,542 prokaryotic genomes, 38 fungal genomes, and 14,224 viral genomes, spanning 786 prokaryotic species, 11 fungal species, and 4,263 vOTUs. Based on the VMGC, analyses have revealed unexplored microbial species, genomic variations, and functional characteristics within the human vaginal microbiome. The VMGC provides a comprehensive resource for studying the vaginal microbiome and its impact on vaginal health. It includes a large number of genomes, with over 25% ofA multi-kingdom collection of 33,804 reference genomes for the human vaginal microbiome has been established, providing a comprehensive resource for studying the vaginal microbiome and its impact on health. The Vaginal Microbial Genome Collection (VMGC) includes 19,542 prokaryotic genomes, 38 fungal genomes, and 14,224 viral genomes, spanning 786 prokaryotic species, 11 fungal species, and 4,263 viral operational taxonomic units (vOTUs). Over 25% of prokaryotic species and 85% of viral vOTUs remain uncultured, highlighting the need for further research. The VMGC significantly enriches genomic diversity, particularly for prevalent vaginal pathogens such as BVAB1 and Amygdalobacter spp. It offers insights into the functional traits of prokaryotes, including the underexplored Saccharofermentales order, and eukaryotic viruses, revealing their niche adaptation and potential roles in the vagina. The VMGC serves as a valuable resource for studying vaginal microbiota and its impact on vaginal health. The human vagina is a diverse ecosystem hosting bacteria, viruses, fungi, and other micro-eukaryotes crucial for maintaining women's and fetal health. In healthy women, Lactobacillus species dominate the vaginal microbiota, producing lactic acid to maintain an acidic environment and prevent harmful microorganisms. Dysbiosis often leads to bacterial vaginosis, characterized by the overgrowth of anaerobic bacteria. Early research relied on culture-based or real-time PCR methods, which had limitations in capturing microbial diversity. Next-generation sequencing has advanced understanding, uncovering uncultivated microorganisms associated with vaginal health. Whole metagenomic sequencing has elucidated the compositional and functional diversity of the vaginal microbiome. The Human Microbiome Project has provided an overview of microbial and functional profiles across various body sites, including the vagina. The study by Jie et al. has uncovered associations between the vaginal microbiota and life history, while Fettweis et al. linked the vaginal microbiota to preterm birth. The VMGC was constructed by integrating available metagenomic datasets and microbial genomes, along with in-house cultivation of vaginal fungi. The VMGC comprises 19,542 prokaryotic genomes, 38 fungal genomes, and 14,224 viral genomes, spanning 786 prokaryotic species, 11 fungal species, and 4,263 vOTUs. Based on the VMGC, analyses have revealed unexplored microbial species, genomic variations, and functional characteristics within the human vaginal microbiome. The VMGC provides a comprehensive resource for studying the vaginal microbiome and its impact on vaginal health. It includes a large number of genomes, with over 25% of