Triptolide (TP), one of the main active ingredients in Tripterygium wilfordii Hook F, is clinically used to treat immune diseases but is known to cause liver injury. The aim of this study was to investigate the biomarkers for TP-induced hepatotoxicity in mice and to determine potential mechanisms of its liver injury. LC/MS-based metabolomics was used to determine the metabolites that were changed in TP-induced liver injury. The accumulation of long-chain acylcarnitines in serum indicated that TP exposure disrupted endogenous peroxisome proliferator-activated receptor alpha (PPAR alpha) signaling. Triptolide-induced liver injury could be alleviated by treatment of mice with the PPAR alpha agonist fenofibrate, whereas the PPAR alpha antagonist GW6471 increased hepatotoxicity. Furthermore, fenofibrate did not protect Ppara-/- mice from TP-induced liver injury, suggesting an essential role for the PPAR alpha in the protective effect of fenofibrate. Elevated long-chain acylcarnitines may protect TP-induced liver injury through activation of the NOTCH-NRF2 pathway as revealed in primary mouse hepatocytes and in vivo. In agreement with these observations in mice, the increase in long-chain acylcarnitines was observed in the serum of patients with cholestatic liver injury compared with healthy volunteers. These data demonstrated the role of PPAR alpha and long-chain acylcarnitines in TP-induced hepatotoxicity, and suggested that modulation of PPAR alpha may protect against drug-induced liver injury.