It has been long recognized that dietary substances can cause significant diet-drug and/or drug-drug interactions. An increased awareness has been given to dietary components that could potentially induce changes of drug disposition via the alteration of drug transporters and metabolizing enzymes. Piperine (PIP), a major component extracted from black pepper, is widely used as daily spice and in some forms of traditional medicine. In the present study, we investigated whether and how piperine can affect the expression of two important genes, CYP3A4 and MDR1, which encode proteins responsible for drug metabolism and transportation. In reporter gene assays using LS74T and LS180 cells, PIP was able to activate PXR-mediated CYP3A4 promoter activity (with an EC50 of 3.6 and 3.9 μM, respectively) and PXR-mediated MDR1 promoter activity (with an EC50 of 1.6 and 3.0 μM, respectively) in a dose-dependent manner. The activity of PIP was blocked by 50 μM of ketoconazole, an inhibitor of PXR activity, further confirming the dependency of PIP’s activity on PXR. In addition, PIP treatment up-regulated endogenous MDR1 protein level in LS180 cell line and endogenous CYP3A4 protein levels in both LS180 and human primary hepatocytes in a dose dependent manner. However, PIP did not directly bind to PXR as revealed in a ligand binding assay and a coactivator recruitment assay. Further study demonstrated that PIP upregulated PXR protein level in LS174T and LS180 cells, providing the first evidence that the induction of CYP3A4 and MDR1 by PIP was through the increased PXR protein level. In view that PXR is a xenobiotc sensor with broad function on genes responsible for drug transportation and metabolism, our results suggest that cautions should be taken for PIP consumption during drug treatment in patients, particularly, who favor daily pepper spice or rely on certain pepper remedy.