Prolactin regulatory element binding (PREB) is a transcription factor that regulates prolactin promoter activity in rat anterior pituitary. The PREB protein is not only expressed in the anterior pituitary but also in the pancreas. We have recently reported that in pancreatic beta-cells, PREB regulates the transcription of the insulin gene in response to glucose stimulation. In the current study, we have examined the role of PREB in regulating glucokinase (GK) in pancreatic beta-cells. To analyse the effects of PREB on GK gene transcription, we employed a reporter gene assay. In the cells expressing or with knocked down PREB, GK expression was determined. GK expression was regulated by glucose and cAMP, and both glucose and cAMP stimulated the expression of PREB in a dose-dependent manner. Conversely, overexpression of PREB using a PREB-expressing adenovirus increased the expression of the GK protein. GK enzymatic activity was also significantly increased in the cells that stably expressed PREB. In addition, PREB induced GK promoter activity. Chromatin immunoprecipitation (ChIP) analyses showed that PREB mediated its transcriptional effect by binding to the PREB-responsive cis-element of the GK promoter. Finally, we used siRNA to inhibit PREB expression in cells and demonstrated that the knockdown of PREB attenuated the effects of glucose and cAMP on GK expression. Our data show that in pancreatic -cells, PREB regulates the transcription of the GK gene in response to glucose and cAMP.