Milk protein yield (MPY) is a crucial milk performance of dairy cows, which is closely related to the profits of dairy farms. This study aimed to investigate the metabolic differences between the cows with higher and lower MPY, and clarify the latent roles of key metabolites. Forty multiparous Holstein dairy cows with day in milk of 160.0 (±29.1 SD) d, and parity of 3.3 (±1.5 SD) were selected from a group of 343 cows fed on a same diet with a ratio of forage-to-concentrate at 40:60. Twenty of them were higher in MPY (>1.11 kg/d, higher milk yield and higher milk protein content, HH) and 20 were lower in MPY (<0.87 kg/d, lower milk yield and lower milk protein content, LL). Serum were collected from all the cows to characterize the metabolites using the gas chromatography–time of flight/mass spectrometry method. Partial least squares discriminant analysis (PLS-DA) and the orthogonal PLS-DA demonstrated a clear disparity of metabolite profiles between the 2 groups. A total of 132 metabolites were identified with 18 metabolites notably different between HH and LL (VIP_predictive > 1 and P < 0.05). Pathway analysis and metabolite sets enrichment analysis revealed that the significantly different metabolites were mainly involved in fructose and mannose metabolism, glycerolipid metabolism, galactose metabolism and arginine and proline metabolism. Among the 18 significantly different metabolites, 16 were higher in HH than in LL. Glucoheptonic acid (log₂FC = 8.19, P < 0.01) that is metal chelating agent was only detected in the HH group (n = 15), suggesting its possible pivotal role in the cows with high MPY. Besides, fucose (log₂FC = 3.49, P < 0.01) relating to sugar metabolism, cancer and various diseases, and glutaric acid (log₂FC = 1.71, P < 0.01) concerning about lysine degradation and fatty acid metabolism in the human were also probably vital for the milk protein synthesis. In sum, our results revealed that there were some metabolic discrepancies in dairy cows with different MPY, which were mainly related to sugar, lipid and amino acid metabolism and immunity.