Neurotensin (NT) is a highly expressed gastrointestinal (GI) neuropeptide, which modulates GI motility, secretion and cell growth as well as intestinal inflammation. Since EGF receptor is highly expressed in human colon cancer cells, we sought to examine whether NT stimulation contributes to the EGFR overexpression using nontransformed colonocyte NCM460 cells. The results show that NT treatment caused a significant increase in EGFR protein expression and gene transcription. Pretreatment with MAP kinase pathway inhibitor PD98059 blocked NT-induced EGFR expression. As the EGFR promoter has a functional Egr-1 site, previously shown to mediate its transcription in response to hypoxia, we examined the role of Egr-1 in the NT response. We first show that NT stimulated Egr-1 expression, which can be inhibited by PD98059. We also determined whether NT increases Egr-1 binding to its site within the EGFR promoter. The data indicate that NT enhanced the amount of Egr-1 binding to the EGFR Egr-1 site and that this binding was significantly decreased by PD98059. To verify that Egr-1 mediated NT-induced EGFR transcription, Egr-1 siRNA was used to knock down its expression. The data show that transfection of Egr-1 siRNA significantly inhibited NT-stimulated EGFR transcription. Together, our results suggest that NT can stimulate MAP kinase-mediated Egr-1 and EGFR gene expression in human colonocytes. Our results may be relevant to the mechanisms by which NT participates in the development of colon cancer.