Hepsin, a type-II membrane-associated serine protease, has been implicated in cell growth and developement as well as possible initiation of blood coagulation. Here, we report on the complete nucleotide sequence, functional characterization of key structural features and the promoter of the mouse hepsin gene. The gene has a size of ≈17 kb, and is composed of 12, 13, or 14 exons depending on alternative intron splicings – one in the 5′-UTR and the other two in the second intron. The latter two, which occur in approximately half of the hepsin transcripts, generate a hepsin mRNA species with an extra exon, which is responsible for producing a hepsin isoform with a unique 20-residue sequence inserted in the cytoplasmic portion of hepsin. Most hepsin transcripts have the 5′-UTR intron spliced, and its splicing can occur independently of the other alternative splicings. The transcriptional initiation site was determined to be 636 bp upstream of the first ATG site in a cytidine-rich region. The 5′-flanking region of hepsin up to nucleotide 274 showed a substantial promoter activity in HepG2 cells, with its expression activity sevenfold higher in the presence of the 5′-UTR intron sequence in comparison to that without the intron sequence. The basal promoter region contains potential binding sites for several transcription factors including SP1, AP2, C/EBP, LF-A1, and E box, which may be responsible for ubiquitous, but liver- and kidney-preferred tissue expression of the hepsin gene.