Triptolide (TP), as the main component of Tripterygium Wilfordii (TW), can induce obvious liver injury when exerting the therapeutic effect. However, in our previous study, Catalpol (CAT), the main active ingredient of Rehmannia Glutinosa (RG), was shown to increase the drug clearance rate of TP and to attenuate TP-induced hepatotoxicity. Thus the present study aims to address the roles of phase I and II metabolic enzymes and the nuclear receptors in the detoxification process of TP, to analyze the mechanism of CAT reducing hepatotoxicity. For this purpose, SD rats and human liver cell line L-02 and HepG2 cells were selected, and treated with TP or the combination of TP and CAT in our study. Then the effect of CAT on detoxification of TP was analyzed, and the roles of phase I metabolic enzymes cytochrome P450 3A2/4 (CYP3A2/4) and phase II metabolic enzyme UDP-glucuronosyltransferase 1A6 (UGT1A6) and their related nuclear receptor regulations were evaluated. It was found that TP inhibited the transcription of CYP3A2/4. And through the constitutive androstane receptor (CAR) pathway, CAT not only significantly changed this inhibition and increased the expression of CYP3A2/4 but also increased the expression of CYP2C9, both of which are phase I detoxification enzymes of TP. And with the gene-silenced experiment, it was confirmed that this regulation was CAR-dependent. We also found that CAT could continue to exert a certain protective effect after CAR was silenced, with UGT1A6, the phase II detoxification enzyme of TP, significantly induced. And this was closely related to the enhanced transcriptional regulation of the nuclear factor erythroid 2-related factor 2 (NRF2) pathway. In conclusion, our results reveal that CAT can induce TP’s phase I detoxification enzymes CYP3A2/4 and CYP2C9 through the CAR pathway, and induce TP’s phase II detoxification enzyme UGT1A6 via the NRF2 pathway when CAR is strongly inhibited. And this coordinate regulation of CAT may be an important source of the effect for CAT to increase TP metabolic conversion and reduce TP hepatotoxicity.