Ectopic expression of the gastrin-releasing peptide (GRP) receptor (GRP-R) occurs frequently in human malignancies of the gastrointestinal tract. Owing to paracrine and autocrine interaction with its specific high-affinity ligand 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 chromatin immunoprecipitation assays two cAMP response element (CRE) binding sites that recruited transcription factor CRE binding protein (CREB) to the human GRP-R promoter. Transfection studies with a wild-type 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 upregulation of human GRP-R transcriptional activity, and this overexpression strictly required intact wild-type 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.