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Background: B and K2 vitamins, essential nutrients in host metabolism, can be synthesized by the rumen microbiome in ruminants and subsequently absorbed by the host. However, the B and K2 vitamin biosynthesis by the whole gastrointestinal microbiome and their abundances in different dietary strategies are largely unknown. Here, we reanalyzed our previous large-scale metagenomic data on the gastrointestinal microbiome of seven ruminant species and recruited 17,425 nonredundant microbial genomes from published datasets to gain a comprehensive understanding of the microbe-mediated B and K2 vitamin biosynthesis in ruminants. Results: We identified 1,135,807 genes and 167 enzymes involved in B and K2 vitamin biosynthesis. Our results indicated that the total abundances of B and K2 vitamin biosynthesis were dominant in the stomach microbiome, while the biosynthesis of thiamine, niacin, and pyridoxine was more abundant in the large intestine. By examining 17,425 nonredundant genomes, we identified 2366 high-quality genomes that were predicted to de novo biosynthesize at least one vitamin. Genomic analysis suggested that only 2.7% of these genomes can synthesize five or more vitamins, and nearly half of genomes can synthesize only one vitamin. Moreover, we found that most genomes possessed cobalamin transporters or cobalamin-dependent enzymes to consume cobalamin directly, and only a few microbial genomes possessed a complete cobalamin biosynthesis pathway. Based on these genomic data, we examined the effect of the high-grain (HG) diet on the vitamin biosynthesis of the rumen microbiome of dairy cattle. We revealed that most vitamin biosynthesis was enhanced in the HG group, while only cobalamin synthesis was inhibited in the HG group, indicating that dietary fiber is vital for cobalamin biosynthesis. Conclusions: We primarily provided a gene catalog and 2366 microbial genomes involved in B and K2 vitamin biosynthesis in ruminants. Our findings demonstrated the regional heterogeneity and dietary effect of vitamin biosynthetic potential in the ruminant gastrointestinal microbiome and interpreted the biosynthesis mechanisms of these microbes and their physiological adaptability. This study expands our understanding of microbe-mediated vitamin biosynthesis in ruminants and may provide novel targets for manipulation to improve the production of these essential vitamins. 6cdWEjSDW5ZW7DH8TeMqdw Video abstract. [ABSTRACT FROM AUTHOR]