The decapeptide GnRH is known for its central role in the regulation of the hypothalamo-pituitary-gonadal axis. In addition, it is also known to have local effects within peripheral tissues. The zinc metalloendopeptidase, EC 220.127.116.11 (EP24.15), can cleave GnRH at the Tyr(5)-Gly(6) bond to form the pentapeptide, GnRH-(1-5). The central and peripheral effect of GnRH-(1-5) is different from its parent peptide, GnRH. In the current study, we examined the effect of GnRH-(1-5) on epidermal growth factor receptor (EGFR) phosphorylation and cellular migration. Using the Ishikawa cell line as a model of endometrial cancer, we demonstrate that GnRH-(1-5) stimulates epidermal growth factor release, increases the phosphorylation of EGFR (P < .05) at three tyrosine sites (992, 1045, 1068), and promotes cellular migration. In addition, we also demonstrate that these actions of GnRH-(1-5) are mediated by the orphan G protein-coupled receptor 101 (GPR101). Down-regulation of GPR101 expression blocked the GnRH-(1-5)-mediated release of epidermal growth factor and the subsequent phosphorylation of EGFR and cellular migration. These results suggest that GPR101 is a critical requirement for GnRH-(1-5) transactivation of EGFR in Ishikawa cells.