Relaxin (RLN2) promotes cell migration/invasion, cell growth, and neoangiogenesis through binding to the relaxin receptor RXFP1 in many types of cancers. However, there have been no studies to determine the role of this system in brain tumors, especially in Glioblastoma Multiforme (GB), the most lethal primary brain tumor in adults. GB is a systemic brain disease and aggressively invades brain tissue. In this study, we have identified RXFP1 receptor, but not RLN2, in GB cell lines and primary GB cells from patients. RLN2 treatment resulted in a significant increase in migration of GB cell line and primary GB cells. To determine molecular mechanisms that facilitate RXFP1-mediated migration in GB cells, we employed a pseudopodia assay and 2D LC-MS/MS to investigate the protein composition at cell protrusions (pseudopodia) during GB cell migration. We also observed the expression of known mediators promoting tissue invasion upon RLN2 treatment. We identified PGRMC1, a candidate protein from 2D LC-MS/MS as a novel relaxin target protein in RXFP1-expressing brain tumor cells. RLN2 treatment also caused an increase in cathepsin (cath)-B and -L and enhanced production of as the small Rho-GTPases Rac1 and Cdc42 in GB cells. Collectively, these findings indicate that RXFP1-induced cell migration is mediated by the upregulation and intracellular actions of Rac1, Cdc42 and by cath-B and cath–L who serve as matrix modulating factors to facilitate brain tumor cells migration. PGRMC1 also contributes to RXFP1-mediated cell migration through an as yet unknown mechanism. RLN2 is not present in the brain. We determined the role of a peptide ligand of RXFP1, the newly discovered C1q/TNF related peptide (CTRP)8-derived P74 peptide, in promoting migration in GB cells. Similar to relaxin, P74 was found to have pro-migratory effects on GB cells. The biological activity of this peptide was also similar to relaxin and caused the upregulation of cath-B, cath-D and cath-L in the primary GB cells, thus, indicating that P74 might serve as a novel RXFP1 activating peptide ligand. We conclude that RXFP1 receptor signaling plays a key role in brain tumors cell migration and invasion.