T-2 toxin is a common contaminant in grains and animal feedstuff, which becomes an increasing threat to human and animal health due to its high toxicity. Investigating the systemic effects of T-2 toxin is important to evaluate the toxicity and facilitate the assessment of food safety. In our investigation, rats were treated with a single dose of T-2 toxin at dosage levels of 0, 0.5, 2.0 and 4.0 mg kg(-1) body weight via gavage. The metabolic profiles of body fluids and multiple organs were obtained by NMR spectroscopy and analyzed by multivariate data analysis methods. The results showed that low and moderate doses of T-2 toxin only influenced the urinary metabonomes, while a high dose of T-2 toxin induced metabolic alterations in urine and multiple organs. These changes included alterations in the levels of membrane metabolites, TCA cycle intermediates, a range of amino acids, nucleosides and nucleotides. T-2 toxin exposure impaired spleen function, causing immunotoxicity, and inhibited protein and DNA biosynthesis. In addition, T-2 toxin also caused oxidative stress and disturbance in energy metabolism and gut microbiome. Our work provided a comprehensive insight into T-2 toxicity and revealed the great potential of metabonomics in assessing the impact of a toxic compound.