The actin cytoskeleton serves as a barrier that protects mammalian cells from environmental pathogens such as bacteria, fungi, and viruses. Several components of antimicrobial signaling pathways have been shown to associate directly with the actin cytoskeleton, indicating that the cytoskeleton may also serve as a platform for immune-associated molecules. Here we report that retinoic acid-induced gene-I (RIG-I), an important viral RNA recognition molecule, is associated with the actin cytoskeleton and localizes predominantly to actin-enriched membrane ruffles in non-polarized epithelial cells. Subcellular localization and fractionation experiments revealed that the association between RIG-I and the actin cytoskeleton was mediated by its N-terminal caspase activation and recruitment domains (CARDs), which were necessary and sufficient to induce cytoskeletal association. We also show that RIG-I plays a role in cellular migration, as ectopic expression of RIG-I enhanced cellular migration in a wound healing assay and depletion of endogenous RIG-I significantly reduced wound healing. We further show that in both cultured intestinal epithelial cells (IEC) and human colon and small intestine biopsies, RIG-I is enriched at apico-lateral cell junctions and colocalizes with markers of the tight junction. Depolymerization of the actin cytoskeleton in polarized IEC led to the rapid relocalization of RIG-I and to the induction of type I interferon signaling. These data provide evidence that RIG-I is associated with the actin cytoskeleton in non-polarized epithelial cells and with the junctional complex in polarized IECs and human intestine and colon biopsies and may point to a physiological role for RIG-I in the regulation of cellular migration.