Urea has been used in diets of dairy cow as a non-protein nitrogen source. It is rapidly hydrolyzed to ammonia which can be used for microbial protein synthesis, but excess ammonia absorbed into blood may be harmful to the animals. However, the changes that occur in the rumen microbial and host blood metabolites after urea nitrogen uptake have not been fully characterized. The objective of this study was to identify changes in rumen microbial and plasma metabolite profiles in dairy cows induced by urea nitrogen using a metabolomics approach. Six dairy cows (550 ± 50 kg BW and 100 ± 21 d in milk) with rumen fistulas were randomly assigned to 2 groups used in a 2 period crossover trial and each experimental period lasted 21 d. All the cows were fed the same total mixed rations, but were intraruminally supplemented with 180 g urea per cow daily or not during the experimental period. Rumen fluid and blood samples were collected and analyzed using nuclear magnetic resonance spectroscopy and multivariate ANOVA. Differences in rumen and plasma metabolite concentrations in cows from the 2 groups were assessed using orthogonal partial least-squares discriminant analysis and identified by searching against related databases. Levels of valine, aspartate, glutamate, and uracil in the rumen, and urea and pyroglutamate in the plasma, were higher (1.36- to 3.17-fold, P < 0.05) in the urea-supplemented group than in the control group. Metabolic pathway analysis of the affected metabolites revealed that pantothenate and CoA biosynthesis, β-alanine metabolism, valine, leucine, and isoleucine metabolism in the rumen, and urea and glutathione metabolism in the plasma were significantly influenced by urea nitrogen. The levels of aspartate and glutamate in the rumen all correlated strongly (r = 0.73 and r = 0.74, respectively, P < 0.01) with the level of urea in plasma. These findings provided novel information to aid understanding of the metabolic pathways affected by urea nitrogen in dairy cows, and could potentially help to guide efforts directed at improving the efficiency of urea utilization in the rumen.