PNRC (Proline-rich Nuclear Receptor Coactivator) is a novel coactivator for multiple nuclear receptors. PNRC was previously identified using bovine SF-1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. To understand the molecular mechanisms that regulate the expression of human PNRC gene, in this study, functional analysis of the 5\' flanking region of the human PNRC gene revealed that the -123/+27 region is the minimal promoter of the human PNRC gene. Gel shift and ChIP analyses demonstrated the specific binding of RFX1 (Regulatory Factor X) protein to the human PNRC promoter region. In co-transfection experiments RFX1 was shown to repress promoter activity of PNRC gene in a dose-dependent manner. These results indicate that r RFX1 specifically bind to promoter region and negatively regulate the transcription of the human PNRC gene.