Glucagon-like peptide-1 and its potent agonist exendin-4 induce several immediate early response genes (IEGs) which code for transcription factors implicated in cell proliferation, differentiation and apoptosis. We recently observed that early growth response factor-1 (EGR-1), one of IEGs products, was required for transcriptional activation of Ccnd1 (cyclin D1) gene by exendin-4. Herein, the regulatory mechanism whereby exendin-4 activates the transcription of EGR-1 gene was investigated in a pancreatic beta-cell line INS-1. Deletion analysis of rat EGR-1 promoter identified a critical region between -73 and -46 for the activation of the EGR-1 gene in response to exendin-4. Mutation of the proximal putative cAMP response element (CRE, 5’-GTACGTCA-3’) located at -69 resulted in a significant decrease in the EGR-1 transcription, whereas the mutation of the distal putative CRE at -139 was without such an effect. In immune supershift assays using exendin-4-treated cells, binding of cAMP responsive element binding protein (CREB) phosphorylated on Ser 133 to the proximal CRE was increased. Employment of a CREB mutant containing Ala substitution at Ser 133 or a dominant negative CREB mutant which inhibits the binding of endogenous CREB to DNA significantly decreased the exendin-4-induced EGR-1 transcription. In experiments using specific protein kinase inhibitors, the effect of H-89 was more prominent than PD98059 indicating the predominance of the PKA signaling over the MEK/ERK in induction of EGR-1 gene. Therefore, it appears that the proximal CRE site is critical and the binding with CREB phosphorylated on Ser 133 is necessary for the induction of EGR-1 transcription by exendin-4.