The constitutive androstane receptor (CAR) is an orphan nuclear receptor which has been shown to participate in the activation of human CYP3A4, which metabolizes more than 50% of clinically used drugs. We investigated the effects of an array of compounds isolated from herbal medicines such as Rheum palmatum (Da Huang), Peucedanum praeruptorum Dunn (Qian Hu), Cortex Mori Radicis (Sang Bai Pi), Radix Asteris (Zi Wan), Salvia miltiorrhiza (Dan Shen), Polygonum cuspidatum Sieb. et Zucc (Hu Zhang), and Ginkgo biloba (Yin Xing) on the CAR-mediated transactivation of CYP3A4. The effect of herbalcompounds on CYP3A4 expression was measured using a CYP3A4 luciferase reporter gene assay in transiently transfected human intestinal LS174T cells. The gene expression, protein expression, and catalytic activity of CYP3A4 in LS174T cells transfected with CAR were determined by using real-time PCR, Western blot analysis, and LC-MS/MS-based substrate assay. The study found that in CAR-transfected cells, praeruptorin A, C, and D significantly induced CYP3A4luciferase activity, mRNA expression, and functional activity through the CAR-mediated pathway; conversely, induction was not found in untransfected cells. Our findings suggest that these herbal compounds can significantly up-regulate the CYP3A4 gene via the CAR-mediated pathway, which has important implications in herb-drug interactions.