Necrotizing enterocolitis (NEC) is associated with the release of interferon-gamma (IFN) by enterocytes and delayed intestinal restitution. Our laboratory has recently demonstrated that IFN inhibits enterocyte migration by impairing enterocyte gap junctions, intercellular channels that are composed of connexin43 (Cx43) monomers and that are required for enterocyte migration to occur. The mechanisms by which IFN inhibits gap junctions are incompletely understood. Lipid rafts are cholesterol-sphingolipid-rich microdomains of the plasma membrane that play a central role in the trafficking and signaling of various proteins. We now hypothesize that Cx43 is present on enterocyte lipid rafts and that IFN inhibits enterocyte migration by displacing Cx43 from lipid rafts in enterocytes. We now confirm our previous observations that intestinal restitution is impaired in NEC and demonstrate that Cx43 is present on lipid rafts in IEC-6 enterocytes. We show that lipid rafts are required for enterocyte migration, that IFN displaces Cx43 from lipid rafts, and that the phorbol ester phorbol 12-myristate 13-acetate (PMA) restores Cx43 to lipid rafts after treatment with IFN in a protein kinase C-dependent manner. IFN also reversibly decreased the phosphorylation of Cx43 on lipid rafts, which was restored by PMA. Strikingly, restoration of Cx43 to lipid rafts by PMA or by transfection of enterocytes with adenoviruses expressing wild-type Cx43 but not mutant Cx43 is associated with the restoration of enterocyte migration after IFN treatment. Taken together, these findings suggest an important role for lipid raft-Cx43 interactions in the regulation of enterocyte migration during exposure to IFN, such as NEC.