Abstract: Objective To construct the cytochrome P-450 (CYP) 3A4 luciferase reporters and test their transcription activity in the positive tumor cells expressed by pregnane X receptor (PXR). Methods The sequences of XREM (-7836/-7208) and PXRE (-362/+52) in CYP 3A4 promoter were amplified via PCR method, and then they were cloned into pGL3-Promoter vectors. The activity of CYP 3A4 reporters was measured by luciferase assay. Results The Rifampicin, which was the agonist of PXR, induced the activity of XREM-Luc (R²=0.92; P=0.003 4) and PXRE-Luc (R²=0.95; P=0.011) in a dose dependent manner, with the EC₅₀value of 5.65±0.58 μmol/L and 8.91±1.12 μmol/L, respectively. The Ketoconazole, which was the antagonist of PXR, inhibited the activity of XREM-Luc (R²=0.92; P=0.003 4) and PXRE-Luc (R²=0.92; P=0.003 4) induced by Rifampicin in a dose dependent manner, with the IC₅₀value of 0.55±0.08 μmol/L and 0.94±0.14 μmol/L, respectively. In addition, the effect of some potential PXR's ligands on CYP 3A4 reporters could be examined in the cells expressed by PXR. Conclusion The construction of the CYP 3A4 reporters is successfully established, which lays foundation for further functional study of CYP 3A4.