Background & Aims: miR-122 is the most abundant microRNA in the liver and regulates metabolic pathways including cholesterol biosynthesis, fatty acid synthesis, and oxidation. However, little is known about mechanisms that regulate the expression of miR-122 in the liver. The aim of this study was to identify key transcriptional regulators for miR-122 expression through intensively studying its primary transcript and promoter region. Methods: Bioinformatics analysis, Northern blotting, RT-PCR, and 5'/3' RACE were performed to analyze miR-122 primary transcript structure, its promoter region, and potential transacting factor binding sites. Reporter gene assays integrated with truncation and site-mutation in miR-122 promoter were performed to determine the trans-activation effect of HNF4 alpha to miR-122-promoter in vitro. ChIP and EMSA assays were performed to determine HNF4 alpha binding to miR-122 promoter. Finally, forced expression and RNAi were performed to verify the regulatory roles of HNF4 to miR-122 expression in vitro and in vivo. Results: Here, we show that miR-122 is processed from a long spliced primary transcript directed by a distal upstream promoter region conserved across species. We dissected this promoter region and identified putative binding sites for liver-enriched transcriptional factors that contribute to the regulation of miR-122 expression, including a putative binding site for hepatocyte nuclear factor 4 alpha (HNF4 alpha). We demonstrate that HNF4 alpha binds to the miR-122 promoter region through the conserved DR-I element. We observed the DR-1-element-dependent activation effect of HNF4 alpha on the conserved miR-122 promoter and the activation could be further enhanced by the addition of PGC1 alpha. Using overexpression and knockdown strategies, we show that HNF4 alpha positively regulates miR-122 expression in both Huh7 cells and the mouse liver. Conclusions: Our results suggest that HNF4 alpha is a key regulator of miR-122 expression in the liver. 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.