This study sought to identify changes in the metabolic signature of arteriovenous plasma metabolome and their changes in response to 3 forage diets [23% of alfalfa hay (AH, n = 10), 30% of corn stover (CS, n = 10), and 30% of rice straw (RS, n = 10), % of DM basis] of 30 Holstein dairy cows (milk yield = 30.0 ± 3.53 kg/d, day in milk = 160 ± 27.8 d, parity = 3.4 ± 1.57; mean ± SD). Diets were isonitrogenous but not isoenergetic, with 31.1 and 18.9%, 36.3 and 19.5%, 36.9 and 21.9% for NDF and ADF in diets of AH, CS, and RS, respectively. The GC-TOF/MS analysis was performed. Multivariate statistical analysis with orthogonal partial least-square discriminant analysis was used. If P-value <0.05, with log₂(fold change) > 0.2 or < −0.2 and q-value <0.15 as cut-off, the metabolites were considered as different. The metabolomics profiling identified a total of 216 and 219 metabolites in the arterial and venous plasma, respectively. Among them, 1 and 4 metabolites were unique to arterial and venous plasma with 25 of them having significantly different abundance between arterial and venous plasma. The KEGG pathway analysis revealed the differential abundant metabolites mainly involved in nutritional transport, amino acid metabolism, and protein translation. Moreover, pathways of aminoacyl-tRNA biosynthesis, phenylalanine metabolism involving glutamate, phenylalanine, and tyrosine, could be selected as the actual pathways to reflect the metabolic progress for milk synthesis. In addition, the significant changes of phenylpropanoate in cows fed CS and RS and prostaglandin A2 in cows fed RS compared with AH, suggesting they are probable biomarker when cows fed low quality forage. The comprehensive assessment of metabolic changes in the arteriovenous metabolome during lactating and under different diets provide some new and fundamental understandings of the important metabolic pathways such as phenylalanine metabolism and aminoacyl-tRNA biosynthesis, and key metabolites such as 3-hydroxybutyric acid, glutamate, phenylpropanoate, and prostaglandin A2 and for improving milk production.