The human HFE gene is clearly involved in hereditary hemochromatosis, a common autosomal recessive genetic disorder of iron homeostasis. However, the precise role of the HFE protein is still undefined. Here, to obtain new insight, we analyzed the transcriptional regulation of HFE gene and defined the functional organization of the HFE promoter. Both in vitro transcription and reporter gene assay in transient transfection evidenced a high liver expression of the HFE mRNA. The 5' end analysis of mRNA showed two major initiation sites localized at -265 and -195 directed by TATA-like sequences and a window of initiation within the -120 to -10 GC-rich region upstream of the first coding nucleotide. Positive cis-regulating elements were characterized within the -1057/-8 region, and a negative one extending upstream (-1485/-1057) was identified. DNase I footprinting analysis and gel shift assay revealed several protein binding sites, and subsequent functional analysis evidenced transactivation of HFE by liver-enriched C/EBPalpha, erythropoietic-specific GATA-1, and ubiquitous Sp1 transcription factors. These data bring some evidence of a role of HFE in the liver and a coregulation with erythropoiesis as other genes involved in iron homeostasis.