Intestinal epithelial cells (IECs) produce several potent cytokines in response to interleukin-1 (IL-1) and may play a role in the inflammatory response. Previously, we determined that treatment of the Caco-2 cells with a cross-linking anti-α3 integrin antibody resulted in a suppression of IL-1 induced cytokine secretion and mRNA levels, suggesting that the α3β1 integrin may play a role in the regulation of IEC cytokine responses to IL-1. In this report, treatment of the Caco-2 cells with the anti-α3 integrin antibody resulted in a suppression of IL-1 induced levels of NF-κB binding activity in nuclear extracts, as determined by EMSA, as well as phosphorylation and degradation of the inhibitor, IκBα. The anti-integrin antibody treatment was also found to suppress IκB kinase (IKK) activity and IKKβ phosphorylation. Culture of the Caco-2 cells on purified laminin-5, the ligand for the α3β1 integrin, also resulted in suppression of IL-1 induced phosphorylation of IκBα and IKKβ. Together with our previous findings, these results suggest that α3β1 integrin binding results in a suppression of the IL-1 signaling pathway leading to the activation of NF-κB and ultimately IEC cytokine responses. These studies define a novel regulatory mechanism which may be important in the control of IEC cytokine responses during inflammation. © 2004 Elsevier Inc. All rights reserved.