Abstract: Ritonavir (RTV) is widely used as an antiviral drug in clinical practice, but the mechanism underlying its hepatotoxicity remains incompletely understood. This study aimed to investigate the molecular mechanism by which pregnane X receptor (PXR) activation exacerbates RTV-induced hepatotoxicity. This study was approved by the Life Science Ethics Review Committee of Zhengzhou University (approval No.: ZZUIRB2022-142). Pxr gene knockout mice were constructed and treated with a PXR agonist and RTV, followed by evaluation of liver function indicators, hepatic pathological changes, expression of the metabolic enzyme, endoplasmic reticulum (ER) stress- and apoptosis-related genes, as well as the major active metabolites of RTV. Cellular experiments were further conducted for validation. The results demonstrated that PXR activation aggravated RTV-induced liver injury in mice, accompanied by upregulation of the relevant metabolic enzyme, accumulation of active RTV metabolites in the liver, elevated ER stress markers, and increased levels of molecules associated with cell death and tissue damage. In HepG2 cells, knockdown/overexpression of PXR respectively inhibited or enhanced rifampicin (RIF, a human PXR agonist)-induced expression of cytochrome P450 (CYP) 3A4. PXR overexpression combined with RIF increased the hepatotoxicity caused by RTV, which was reversed after CYP3A4 knockdown. Mechanistically, PXR activation accelerates the metabolism of RTV into toxic metabolites via upregulating CYP3A4, and these metabolites trigger ER stress, thereby promoting hepatotoxicity. This study reveals the central role of the PXR-CYP3A4 axis in RTV-induced hepatotoxicity by triggering ER stress, providing a novel strategy for mitigating drug-induced liver injury through targeted modulation of PXR.