CYP2A6 plays important roles in the metabolism of nicotine and some clinically used drugs. Interindividual variability in the CYP2A6 expression level in human liver might be caused by an inducible property, but the molecular mechanism of induction is unclear. Rifampicin, phenobarbital, and 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime, which are activators of pregnane X receptor (PXR) and constitutive androstane receptor (CAR), induced CYP2A6 mRNA in human hepatocytes. We identified three direct repeat separated by four nucleotides (DR4)-like elements at -6698, -5476, and -4618 in the CYP2A6 gene, to which PXR and CAR could bind after dimerization with retinoid X receptor (RXR)-alpha. In luciferase assays, overexpression of PXR or CAR could not activate the transcriptional activity of CYP2A6 promoter constructs (-6754 to -1) in HepG2 cells. Cotransfection of hepatocyte nuclear factor-4alpha did not affect the transcriptional activities in the absence or presence of PXR or CAR. Interestingly, cotransfection of peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha) as well as PXR significantly enhanced the transcriptional activity (3.9-fold of control). By the deletion of a possible suppresser region (-4533 to -185), the effects of PXR/PGC-1alpha on the transcriptional activity were increased (6.9-fold of control). Deletion or mutation analyses revealed that two DR4-like elements at -5476 and -4618 are essential for transactivation by PXR/PGC-1alpha. Chromatin immunoprecipitation assay revealed that PXR and PGC-1alpha bind to CYP2A6 chromatin. In conclusion, we found that CYP2A6 is induced via PXR and PGC-1alpha through the DR4-like element at the distal response region. This is the first study to report the molecular mechanism of the induction of CYP2A6.