Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. Vaccines require ∼7 days to induce protection; thus, before this time, vaccinated animals are still susceptible to the disease. Our group has previously shown that swine inoculated with 1×10(11) focus forming units (FFU) of a replication-defective human adenovirus containing the gene for porcine interferon alpha (Adt-pIFN-α) are sterilely protected from FMDV serotypes A24, O1 Manisa, or Asia 1 when the animals are challenged 1 day postadministration, and protection can last for 3-5 days. Polyriboinosinic-polyribocytidylic acid stabilized with poly-l-lysine and carboxymethyl cellulose (poly ICLC) is a synthetic double-stranded RNA that is a viral mimic and activates multiple innate immune pathways through interaction with toll-like receptor 3 and MDA-5. It is a potent inducer of IFNs. In this study, we initially examined the effect of poly IC and IFN-α on FMDV replication and gene induction in cell culture. Poly ICLC alone or combined with Adt-pIFN-α was then evaluated for its therapeutic efficacy in swine against intradermal challenge with FMDV A24, 1 day post-treatment. Groups of swine were subcutaneously inoculated either with poly ICLC alone (4 or 8 mg) or in combination with different doses of Adt-pIFN-α (2.5×10(9), 1×10(9), or 2.5×10(8) FFU). While different degrees of protection were achieved in all the treated animals, a dose of 8 mg of poly ICLC alone or combined with 1×10(9) FFU of Adt-pIFN-α was sufficient to sterilely protect swine when challenged 24 h later with FMDV A24. IFN-stimulated gene (ISG) expression in peripheral blood mononuclear cells at 1 day post-treatment was broader and higher in protected animals than in nonprotected animals. These data indicate that poly ICLC is a potent stimulator of IFN and ISGs in swine and at an adequate dose is sufficient to induce complete protection against FMD.