Although early growth response-1 (EGR-1) has been shown as a key transcription factor in controlling cell growth, proliferation, differentiation, and angiogenesis, its role in the development of esophageal cancer is poorly understood despite the high frequency of this disease in many parts of the world. Here, immunohistochemistry showed that EGR-1 is overexpressed in 80% of esophageal tumor tissues examined. Furthermore, EGR-1 is constitutively expressed in all esophageal cancer cell lines analyzed. Esophageal squamous carcinoma WHCO1 cells stably transfected with EGR-1 short hairpin RNA displayed a 55% reduction in EGR-1 protein levels, 50% reduction in cell proliferation, a 50% reduction in cyclin-dependent kinase 4 levels, and a 2-fold induction in p27Kip1 levels associated with a G2-M cell cycle arrest. EGR-1 knockdown also caused a marked induction in IκBα expression, an effect also observed in GROβ RNA interference-expressing WHCO1 cells, because EGR-1 lies downstream of GRO/CXCR2 signaling. Furthermore, p65 mRNA levels were also reduced in cells treated with either short hairpin RNA EGR-1 or small interfering RNA EGR-1. Immunohistochemical analysis indicated that p65 is elevated in 78% (n = 61) of esophageal tumor sections analyzed. Moreover, nuclear factor-κB inhibition with either sodium salicylate or p65 RNA interference led to a significant reduction in GROα and GROβ expression. These results indicate that EGR-1 and nuclear factor-κB mediate GRO/CXCR2 proliferative signaling in esophageal cancer and may represent potential target molecules for therapeutic intervention. Copyright © 2009 American Association for Cancer Research.