The cAMP signaling pathway stimulates cholecystokinin (CCK) gene transcription via CREB binding to a cAMP response element (CRE). In the present study we examined the function of glycogen synthase kinase-3beta (GSK-3beta) on cAMP-induced CCK gene transcription. Co-transfection of GSK-3beta reduced the cAMP-induced CCK promoter activity with approximately 80% and approximately 60% in SK-N-MC and PC12 cells, respectively. Binding of in vitro translated CREB to the CCK CRE(-80) promoter element was reduced following incubation with recombinant GSK-3beta. Finally, mutation of serine-129, which is a phosphorylation site for GSK-3beta, did not abolish cAMP-induced CREB-dependent transcription, and cAMP-mediated GAL4-CREB transcription was unaffected by co-transfection with GSK-3beta. We conclude that GSK-3beta regulates cAMP-induced CCK transcription by reducing CREB binding to the CRE(-80) element in the CCK promoter.