Epithelial restitution, the movement of wound-edge cells into an area of epithelial cell denudation, is an important early step in the ulcer healing process. Growth factors regulate epithelial restitution, yet little is known about the transcriptional pathways that mediate their effects on cell migration. The transcription factor nuclear factor (NF)-κB is a master regulator of the host inflammatory response that is activated in the epithelium in intestinal inflammation, which often accompanies epithelial injury. We hypothesized that NF-κB may be an important transcriptional regulator of epithelial restitution. In an in vitro model of scrape-wounded monolayers of nontransformed rat intestinal epithelial (RIE-1) cells, NF-κB was activated in epithelial cells at the wound edge. Blocking of NF-κB activation by either pharmacological or genetic approaches inhibited intestinal epithelial restitution. Moreover, scrape wounding activated the epidermal growth factor receptor (EGFR) in cells at the wound edge, and, importantly, inhibiting EGFR tyrosine kinase activity decreased scrape wound-induced NF-κB activation and cell migration. These results indicate a novel role of NF-κB activation in a signaling pathway important for restitution and healing of intestinal epithelia. To the extent NF-κB may have parallel functions in vivo, they also suggest a need for caution in the proposed use of NF-κB inhibitors for the treatment of conditions associated with inflammation and injury of intestinal and other mucosal surfaces.