Vitamin B 12 is synthesized by prokaryotes in the rumens of dairy cows-and this has implications in human nutrition since humans rely on consumption of dairy products for vitamin B 12 acquisition. However, the concentration of vitamin B 12 in milk is highly variable, and there is interest in determining what causes vitamin B 12 variability. We collected 92 temporally linked rumen, fecal, blood, and milk sample sets from Holstein cows at various stages of lactation fitted with rumen cannula and attempted to define which bacterial genera correlated well with vitamin B 12 abundance. The level of vitamin B 12 present in each sample was measured, and the bacterial population of each rumen, fecal, and milk sample (n ϭ 263) was analyzed by 16S rRNA-targeted amplicon sequencing of the V4 region. The bacterial populations present in the rumen, small intestine, and milk were highly dissimilar. Combined diet and lactation status had significant effects on the composition of the microbiota in the rumen and in the feces. A high ruminal concentration of vitamin B 12 was correlated with the increased abundance of Prevotella, while a low ruminal concentration of vitamin B 12 was correlated with increased abundance of Bacteroidetes, Ruminiclostridium, and Butyrivibrio. The ultimate concentration of vitamin B 12 is controlled by the complex interaction of several factors, including the composition of the microbiota. Bacterial consumption of vitamin B 12 in the rumen may be more important in determining overall levels than bacterial production. IMPORTANCE In this paper, we examined the microbiome of the bovine rumen, feces, and milk and attempted to understand how the bacterial communities at each site affected the production and movement of vitamin B 12 around the animal's body. It was determined that the composition of the bovine rumen microbiome correlates well with vitamin B 12 concentration, indicating that the rumen microbiota may be a good target for manipulation to improve production of this important vitamin.