HNF1α is a transcription factor that is expressed in pancreatic β-cells and mutations of the HNF1α gene cause a form of monogenic diabetes. To understand the role of HNF1α in pancreatic β-cells, we established the MIN6 β-cell line that stably expressed HNF1α-specific shRNA. Expression of the gene encoding hepatocyte growth factor (HGF) activator (Hgfac), a serine protease that efficiently activates HGF, was decreased in HNF1α KD-MIN6 cells. Down-regulation of Hgfac expression was also found in the islets of HNF1α (+/-) mice. Reporter gene analysis and the chromatin immunoprecipitation assay indicated that HNF1α directly regulates the expression of Hgfac in β-cells. It has been reported that HGF has an important influence on β-cell mass and β-cell function. Thus, HNF1α might regulate β-cell mass or function at least partly by modulating Hgfac expression.