Human cytochrome P450 2A6 (CYP2A6) metabolizes various clinically relevant compounds, including nicotine-and tobaccospecific procarcinogens; however, transcriptional regulation of this gene is poorly understood. We investigated the role of the glucocorticoid receptor (GR) in transcriptional regulation of CYP2A6. Dexamethasone (DEX) increased CYP2A6 mRNA and protein levels in human hepatocytes in primary culture. This effect was attenuated by the GR receptor antagonist mifepristone (RU486; 17␤-hydroxy-11␤-[4-dimethylamino phenyl]-17␣-[1-propynyl]estra-4,9-dien-3-one), suggesting that induction of CYP2A6 by DEX was mediated by the GR. In gene reporter assays, DEX caused dose-dependent increases in luciferase activity that was also prevented by RU486 and progressive truncations of the CYP2A6 promoter delineated DEX-responsiveness to a Ϫ95 to ϩ12 region containing an hepatic nuclear factor 4 (HNF4) ␣ response element (HNF4-RE). Mutation of the HNF4-RE abrogated HNF4␣-and DEX-mediated transactivation of CYP2A6. In addition, overexpression of HNF4␣ increased CYP2A6 transcriptional activity by 3-fold. DEX increased HNF4␣ mRNA levels by 4-fold; however, the amount of HNF4␣ nuclear protein was unaltered. Electrophoretic mobility shift, chromatin immunoprecipitation (ChIP), and streptavidin DNA binding assays revealed that DEX increased binding of HNF4␣ to the HNF4-RE and that an interaction of GR and HNF4␣ occurred at this site. Moreover, ChIP assays indicated that histone H4 acetylation of the CYP2A6 proximal promoter chromatin was increased by DEX that may allow for increased binding of HNF4␣ to the HNF4-RE in human hepatocytes. These findings indicate that increased expression of CYP2A6 by DEX is mediated by the GR via a nonconventional transcriptional mechanism involving interaction of HNF4␣ with an HNF4-RE rather than a glucocorticoid response element.