There are three enzymes involved in the biosynthesis of the adrenal androgen dehydroepiandrosterone sulfate (DHEAS). Cholesterol side-chain cleavage (CYP11A1) and 17-hydroxylase/ 17,20-lyase (CYP17) metabolize cholesterol into dehydroepiandrosterone (DHEA), while steroid sulfotransferase family 2A1 (SULT2A1) is responsible for conversion of DHEA to DHEAS. We previously examined the mechanisms regulating CYP11A1, CYP17, and SULT2A1 transcription and found that each is regulated, in part, by the transcription factor GATA-6. Previous studies have suggested that mediator complex subunit 1 (MED1, also called PPARBP or TRAP220) is a cofactor involved not only in the regulation of nuclear receptors but also in the activation of GATA-6 transcription. Herein, we demonstrated a role for MED1 in the regulation of CYP11A1, CYP17, and SULT2A1 transcription. Transient transfection assays with SULT2A1 deletion and mutation promoter constructs allowed the determination of specific the GATA-6 binding cis-regulatory elements necessary for transactivation of SULT2A1 transcription. Binding of MED1 and GATA-6 was confirmed by co-immunoprecipitation/Western analysis, and chromatin immunoprecipitation (ChIP) assay. We demonstrated expression of MED1 mRNA and protein in the human adrenal, and determined that knockdown of MED1 expression via specific siRNA attenuated CYP11A1, CYP17, and SULT2A1 expression levels in H295R cells. In addition, we demonstrated that MED1 enhanced GATA-6 stimulated transcription of promoter constructs for each of these genes. Moreover, the activity of MED1 for SULT2A1 promoter was mediated by GATA-6 via the -190 GATA-binding site. These data support the hypothesis that MED1 and GATA-6 are key regulators of SULT2A1 expression, and they play important roles in adrenal androgen production.