Ectopic expression of the gastrin-releasing peptide receptor (GRP-R) occurs frequently in human malignancies of the gastrointestinal tract. Owing to paracrine and autocrine interaction with its specific high-affinity ligand gastrin-releasing peptide (GRP), tumor cell proliferation, migration and invasion might ensue. Here we provide the first insights regarding molecular mechanisms of GRP-R regulation in gastrointestinal cancer cells. We identified by EMSA and ChIP assays two cAMP response element (CRE) binding sites that recruited transcription factor CREB to the human GRP-R promoter. Transfection studies with a wildtype human GRP-R promoter reporter and corresponding CRE mutants showed that both CRE sites are critical for basal transcriptional activation in gastrointestinal cancer cells. Forced expression of cAMP-dependent effectors CREB and PKA resulted in robust up-regulation of human GRP-R transcriptional activity and this over-expression strictly required intact wildtype CRE sites. Direct cAMP stimulation with forskolin resulted in enhanced human GRP-R promoter activity only in HuTu 80 cells, but not in Caco-2 cells, coinciding with forskolin-induced CREB phosphorylation occurring only in HuTu 80 but not Caco-2 cells. In summary, CREB is a critical regulator of human GRP-R expression in gastrointestinal cancer and might be activated through different upstream intracellular pathways.