There is a gap in our understanding of mechanisms controlling the B-vitamin status and content in milk despite their primordial nutritional value. Rumen microbes synthesise B vitamins, and there is evidence that vitamins from diet are modified by microbial activity. This work explored the mechanisms regulating B-vitamin post-rumen availability by comparing the composition and activities of rumen microbes in two ruminant species. Four Holstein cows and 4 Alpine goats were enrolled simultaneously in two 4x4 Latin square designs, each consisting of four 4-week periods and 4 dietary treatments. The control diet (CTL, 45%:55% hay:concentrate) was supplemented (% of dry matter intake) with wheat and corn oil (COS), 1.5% algae powder (MAP) or 3% hydrogenated palm oil (HPO). At the end of each period, blood, milk and rumen contents were sampled. Vitamins were analysed by liquid chromatography for B2 and B6 and radioassay for B9 and B12. Metatranscriptomic analysis of rumen contents was performed by mapping mRNA against an in-house database of B-vitamins related genes, while rRNA were used for taxonomic affiliation using Silva. B9 concentrations in plasma and milk and B12 in milk were higher in cows, independent of diet. Accordingly, we observed in cows a higher abundance of Firmicutes, predicted to possess the corresponding biosynthesis pathways. Conversely, goats’ plasma had higher B12 levels, and differential expression analysis highlighted 3 genes related to the cobalamine biosynthesis pathway overexpressed in goats rumen microbiota. Diet did not affect vitamins concentrations in goats; likewise, no significant diet-related changes were observed on goat rumen microbial taxonomy. In cows, the COS diet increased B2 milk concentrations, accompanied by an increase in Bacteroidetes abundance, phylum shown to contain all essential genes for riboflavin biosynthesis. Analysis underway aims to construct a tripartite network integrating gene expression, taxonomy and B vitamins concentrations in plasma and milk.