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An NMR-based metabonomic approach was used to examine rat's urinary response to dosage of triptolide (TP), a major component responsible for the immunosuppressive and anti-inflammatory effects of Tripterygium wilfordii Hook F (TWHF). The urine samples of Wistar rats were collected at various time intervals before and after dosage of TP (i.p.) and measured using conventional 1 H NMR spectroscopy. The data were statistically analyzed using a principle component analysis (PCA). The results showed that biochemical variation induced by TP was time-related, and the maximal alteration in the metabolites appeared at 16 h, and partially recovered 56 h later after dosage. Increment in relative concentrations of taurine, creatine, trimethylamine N-oxide and decrement in citrate, succinate, 2-oxoglutarate and hippurate were observed in the urine after dosage of TP. In addition, 2'-deoxycytidine appeared 0-16 h later after the dosage, which may be considered as another biomarker for the acute hepatotoxicity. It suggested that TP may disturb the metabolism of energy and gut microflora, and may cause acute liver lesion and a slight renal impair. These results were also supported by the conventional analysis of clinical plasma chemistry and histopathology. The information observed in this article may be useful for giving insight into mechanism of liver injury induced by TP.