B vitamins in milk can come from a cow’s diet but also from ruminal synthesis. Recent studies indicate that cows’ diet can have an impact on B vitamin content of milk but it is not known if the vitamin status of the cows is also impacted. This study investigated how the abundance of vitamin B1, B2, B5, B6, B9, and B12 in milk and of potential blood biomarkers for these vitamins vary, as well as the impact of ruminal synthesis. Four groups of 10 dairy cows, received diets differing in the percentage and nature of forage. Individual milk, blood, and ruminal contents were collected after 8 weeks (July sampling) and after 19 weeks (September sampling) on these diets. Samples were analyzed for vitamin content B vitamins. RNA sequencing as well as 16s rRNA gene sequencing were performed on stomach fluid samples from each cow to investigate the functional and taxonomic composition of the microbiota present and determine how this community contributes to vitamin production. Vitamin B2 content in milk was higher when the proportion of grass increased in the diet, but only in July. Vitamin B12 content is higher with a corn-based diet with a little access to pasture. Vitamin B5 content is the highest for corn-based diets without grazing time. Surprisingly, no variation was observed at the blood level. RNA sequencing results show that pathways of B vitamin synthesis are upregulated in the conditions that corresponded to increased B vitamin content. However, some of the variations of the gene activities were not related to variations of B vitamins concentrations in milk or blood. Several hypotheses could explain this. The compounds quantified in blood could be not good biomarkers of B vitamins. B vitamins can also be degraded in the rumen and use or degradation of vitamins in other organs may explain the disconnect between the concentrations in milk or blood. Future investigations of B vitamins production and use by rumen microbiota may help to shed light on the relationships between feed and milk B vitamin concentrations.