Gastrin-releasing peptide (GRP) has been proposed as a major growth factor in brain tumors, and GRP receptor (GRPR) antagonists show antiproliferative effects in experimental gliomas. However, the underlying molecular events downstream of GRPR activation remain poorly understood. In the present study, we examined the role of the GRPR in regulating proliferation of glioma cells in vitro and its possible interaction with the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Expression of GRPR mRNA and protein in C6, U-87MG, and U-373MG glioma cells was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Proliferation of C6 and U-87MG, but not U-373MG cells was significantly inhibited by the GRPR antagonist RC-3095, whereas the GRPR agonist bombesin (BB) significantly enhanced proliferation of C6 cells. The BB-induced stimulatory effect on cell proliferation was prevented by either RC-3095 or the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Our results provide the first evidence that the GRPR regulates proliferation of C6 glioma cells and suggest that PI3K is required for GRPR-mediated stimulation of glioma growth.