The regulation of hepatic glucose metabolism is important in glucose homeostasis, and liver glucokinase (LGK) plays a central role in this process. Hepatic glucokinase expression is known to be regulated by insulin. Recently it has been suggested that sterol regulatory element binding protein-1c (SREBP-1c) mediates the action of insulin on LGK transcription; however, the precise mechanism is not, to date, well known. In the present study, we identified two functional SREBP-1c response elements, SREa and SREb, in the rat LGK promoter. SREBP-1c could bind to these SREs and activate the LGK promoter, and insulin activated the LGK promoter in Alexander cells. The physical interaction between the protein and SREs of the LGK promoter in vivo was also confirmed. Insulin selectively increased SREBP-1c and LGK expression in primary hepatocytes. Adenoviral expression of SREBP-1c stimulated LGK expression, and the dominant negative mutant of SREBP-1c blocked the increased gene expression of LGK by insulin and SREBP-1c. A chromatin immunoprecipitation assay using primary hepatocytes showed increased binding of SREBP-1 to SREs of the LGK promoter by insulin.