Background: Mediator subunit MED25 associates with hepatocyte nuclear factor 4␣ to initiate activation of select genes. Results: CYP2C9 activation by MED25 involves preinitiation complex formation, H3K27 acetylation, and chromatin conformation changes, and the absence of MED25 results in H3K27 trimethylation and Polycomb group recruitment. Conclusion: MED25 is a key regulator of epigenetic mechanisms. Significance: MED25 regulates human drug metabolism gene CYP2C9 by epigenetic modification.