PREB (prolactin regulatory element binding) protein has been identified as a factor that regulates prolactin promoter activity in rat anterior pituitary. PREB is located not only in the anterior pituitary but also in pancreas, however its role in pancreas is not known. Therefore, we examined the role of PREB in insulin gene expression. To analyze the effects of PREB on insulin gene transcription, we employed the reporter gene assay and EMSA. In the cells expressing or knocked down the PREB, insulin expression and secretion were determined. PREB was located mainly in nuclei of rat pancreatic β-cells and its cell line, INS-1. A nuclear extract of INS-1 cells contained material that was recognized by PREB antiserum. Furthermore, this nuclear extract contained insulin promoter binding activity that was super-shifted by PREB antiserum in EMSA studies. In the INS-1 cells, the co-expression of PREB and the insulin promoter induced activity of the latter. The addition of glucose to the cells increased PREB expression. Deletional analysis of the insulin promoter showed that A3, a glucose-responsive cis-element in the insulin promoter mediated the transcriptional effect of PREB. Additionally, synthesized PREB bound the A3 element by EMSA, while a mutant of this motif in the insulin promoter abrogated the effect of PREB. Furthermore, cells expressing or knock-down PREB exhibited increased or decreased insulin expression, respectively. These results demonstrate that PREB may contribute to the regulation of insulin gene transcription and insulin secretion in response to glucose stimulation.