Type III interferons (IFNs) are a family of recently identified antiviral cytokines. One to 3 paralogs have been identified in several species; however, little information is available about type III IFNs in pigs. We have identified 2 porcine type III IFNs, Sus scrofa IFN-λ1 (SsIFN-λ1) and SsIFN-λ3, and determined their tissue expression profile and antiviral activities. Open reading frames of SsIFN-λ1 and SsIFN-λ3 are 576 and 588 bp, encoding 191 and 195 amino acid preproteins, respectively. In healthy pigs, SsIFN-λ3 was primarily expressed in mesenteric lymph nodes and intestine, whereas expression of SsIFN-λ1 was found in all tested tissues and was high in mesenteric lymph nodes, intestine, and liver. Porcine cells treated with the viral mimic, dsRNA, robustly increased SsIFN-λ3 expression, with epithelial cells generally displaying the greatest response. Conversely, dsRNA-induced mRNA expressions of SsIFN-λ1, SsIFN-α1, and SsIFN-β were relatively weaker and delayed compared with SsIFN-λ3. SsIFN-λ1 and SsIFN-λ3 peptides exerted similar but lower antiviral potency than SsIFN-α1 and SsIFN-β against a porcine arterivirus and an adenovirus. These findings indicate that pigs have 2 type III IFN paralogs, which have antiviral activity and may serve as targets for modulation of the porcine host-pathogen interaction.