A novel metabolomic method based on gas chromatography-mass spectrometry was applied to investigate serum metabolites in response to dietary Gln supplementation in piglets. Sixteen, 21-d-old pigs were weaned and assigned randomly to 2 isonitrogenous diets: 1) Gln diet, which contained 1% L-Gln (as-fed basis), and 2) control diet, which contained L-Ala to make this diet isonitrogenous with the Gln diet. Serum samples were collected to characterize metabolites after a 30-d treatment. in addition, 4 liver samples per treatment were collected to examine enzyme activity and gene expression involved in metabolic regulation. Results indicated that 12 metabolites were altered (P < 0.05) by Gln treatment, including carbohydrates, AA, and fatty acids. A leave-one-out cross validation of random forest analysis indicated that Pro was most important among the 12 metabolites. Thus, these data demonstrate that the control and Gln-supplemented pigs differed (P < 0.05) in terms of metabolism of carbohydrates, Pro, Tyr, and glycerophospholipids. Principal component analysis yielded separate clusters of profiles between the Gln and control groups. Metabolic enzyme activities of Ala aminotransferase and hexokinase increased by 26.8% (P = 0.026) and 26.2% (P = 0.004) in the liver of Gln-supplemented pigs vs. control, respectively, whereas pyruvate kinase (PK) activity decreased by 29.1% (P = 0.001). The gene expression of PK in the liver decreased by 66.1% (P = 0.034) by Gln treatment for 30 d. No differences were observed for the mRNA abundance of mammalian target of rapamycin and PPARγ. On the basis of these data, Gln treatment affected carbohydrate, lipid, and AA metabolism in the whole body of the early weaned piglets. These findings provide insight into specific metabolic pathways and lay the groundwork for the complex metabolic alteration in response to dietary Gln supplementation of pigs.