Deoxynivalenol (DON), a Fusarium mycotoxin, causes health hazards for both humans and livestock. Therefore, the aim of this study was to investigate the metabolic profiles of the liver, serum, and urine of piglets fed DON using proton nuclear magnetic resonance (1H NMR) spectroscopy. The 1H-NMR spectra of the liver, serum, and urine samples of the piglets provided with feed containing 8 mg DON/kg for 4 weeks were aligned and identified using the icoshift algorithm of MATLAB R2013b. The data were analyzed by multivariate analysis and by MetaboAnalyst 4.0. The DON-treated groups exhibited discriminating metabolites in the three different sample types. Metabolic profiling by 1H-NMR spectroscopy revealed potential metabolites including lactate, glucose, taurine, alanine, glycine, glutamate, creatine, and glutamine upon mycotoxin exposure (variable importance in the projection, VIP > 1). Forty-six metabolites selected from the principal component analysis (PCA) helped to predict sixty-five pathways in the DON-treated piglets using metabolite sets containing at least two compounds. The DON treatment catalyzed the citrate synthase reactions which led to an increase in the acetate and a decrease in the glucose concentrations. Therefore, our findings suggest that glyceraldehyde-3-phosphate dehydrogenase, citrate synthase, ATP synthase, and pyruvate carboxylase should be considered important in piglets fed DON contaminated feed. Metabolomics analysis could be a powerful method for the discovery of novel indicators underlying mycotoxin treatments.