2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated to have the adverse effects on human health. In this study, we applied a metabolomic approach in conjunction with unsupervised and supervised machine learning methods to investigate the toxic effects of TCDD. By using liquid chromatography/quadrupole time-of-flight mass spectrometry, non-targeted metabolomic analysis revealed the metabolic signatures of the toxicity in aryl hydrocarbon receptor (AhR)-high affinity C57BL/6J (C6) mice as well as low affinity strain-DBA/2J (D2) mice. Lysophospholipids and long chain fatty acids were strikingly elevated in the C6 mice exposed to TCDD in both liver and skeletal muscle tissues. Meanwhile, the level of palmitoylcarnitine, which is one of the important indicators in fatty acid β-oxidation, increased significantly. Moreover, several nucleosides and amino acids decreased markedly. On the other hand, much less differentiating metabolites were highlighted in another strain-D2 mouse model. Taking liver and skeletal muscle tissues together, the levels of inosine, valine and glutamine decreased significantly. One lysophospholipid and two fatty acids were found to be enhanced. The principal components analysis and support vector machine clustering results also exhibited discriminations in the liver and skeletal muscle tissues of the mice. The obtained results indicated that TCDD could disrupt several metabolic pathways, including fatty acid biosynthesis and amino acid metabolism in both C6 and D2 mice. The increased rate of fatty acid beta-oxidation, however, was only observed in the liver and skeletal muscle tissues of C6 mice. The perturbation of the tricarboxylic acid (TCA) cycle was testified in two strains but the change was much slighter in D2 mice. It was of particular interest to note that the succinate level was enhanced in the liver tissues of both strains, and particularly, the change was up to 11.49-fold in the liver of C6 mice treated with TCDD. Collectively, the discrimination of D2 mice was not as distinct as that of C6 mice when exposed to the same dosage. Furthermore, D2 was confirmed to be less-sensitive rather than resistant to a high dose of TCDD.