The CYP2C subfamily of cytochrome P450 enzymes is an important class of drug metabolizing enzymes in human liver. CYP2C9 is the most abundant member of the human CYP2C subfamily in liver and metabolizes ~15% of the therapeutic drugs as well as other xenobiotics and endogenous compounds. A number of nuclear receptors including xenobiotic-sensing receptors such as the constitutive androstane receptor (CAR), pregnane X receptor (PXR), and glucocorticoid receptor (GR) as well as liver enriched receptors hepatic nuclear factor 4α (HNF4α) and the estrogen receptor α (ERα) regulate CYP2C9 expression. Here, we show that Med25, a variable component of Mediator complex, enhanced ligand dependent ERα-mediated transcriptional activation of CYP2C9 promoter and interacts with activated ERα by 17β-estradiol through its C-terminal LXXLL motif. In conclusion, Med25 is identified as a new coactivator of ERα that is required for ERα-mediated regulation of CYP2C9 expression.