Retinoids and carotenoids are frequently used as antioxidants to prevent cancer. In this study, a panel of retinoids and carotenoids was examined to determine their effects on activation of RXR/CAR-mediated pathway and regulation of CYP3A gene expression. Transient transfection assays of HepG2 cells revealed that five out of thirteen studied retinoids significantly induced RXRalpha/CAR-mediated activation of luciferase activity that is driven by the thymidine kinase promoter linked with a PXR binding site in the CYP3A4 gene [tk-(3A4)(3)-Luc reporter]. All-trans retinoic acid (RA) and 9-cis RA were more effective than CAR agonist TCBOPOP in induction of the tk-(3A4)(3)-Luc reporter. Addition of retinoid and TCBOPOP further enhanced the inducibility and the induction was preferentially mediated by RXRalpha/CAR and RXRgamma/CAR heterodimer. Chromatin immunoprecipitation assay showed that retinoids recruit RXRalpha and CAR to the proximal ER6 and distal XREM nuclear receptor response elements of the CYP3A4 gene promoter. The experimental data demonstrate that retinoids can effectively regulate CYP3A gene expression through the RXR/CAR-mediated pathway.