Triptolide (TP) is an active component of Tripterygium wilfordii Hook. F and widely used to treat autoimmune and inflammatory diseases. It has been demonstrated that cytochrome P450 (CYP) are involved in the metabolism of TP. However, the underlying mechanisms of TP-induced toxicity mediated by hepatic CYP have not been well delineated. In this study, rat liver microsomes (RLM) and sandwich-cultured rat hepatocytes (SCRH) were used to identify the mechanism involving the CYP3A inhibition by TP and to evaluate TP-induced liver damage after CYP3A modulation by the known inhibitor, ketoconazole, and the known inducer, dexamethasone. The results showed that TP itself had a time- and concentration-dependent inhibitory effect on CYP3A. When the CYP3A inhibitor and inducer were added, the enzyme activity and hepatotoxicity changed significantly. The enzyme inducer increased CYP3A activity and decreased the metabolic half life (t_1/2) of TP when compared to the control group, while the enzyme inhibitor had an opposite effect. Our findings reveal that TP is a weak CYP3A inhibitor involving the time-dependent inhibition mechanism. The induction or inhibition of CYP3A played an important role in TP-induced hepatotoxicity. Clinicians should be aware of the metabolic characteristics of TP to maximize therapeutic efficacy and reduce TP-induced toxicity.