IFNs play a critical role in innate immunity against viral infections. Melanoma differentiation-associated protein 5 (MDA5), an RNA helicase, is a key component in activating the expression of type I IFNs in response to certain types of viral infection. MDA5 senses noncellular RNA and triggers the signaling cascade that leads to IFN production. Synthetic double-stranded RNAs are known activators of MDA5. Natural single-stranded RNAs have not been reported to activate MDA5, however. We have serendipitously identified a viral mRNA from parainfluenza virus 5 (PIV5) that activates IFN expression through MDA5. We provide evidence that the signaling pathway includes the antiviral enzyme RNase L. The L mRNA of PIV5 activated expression of IFN-β. We have mapped the RNA to a region of 430 nucleotides within the L mRNA of PIV5. Our results indicate that a viral mRNA, with 5'-cap and 3'-poly (A), can activate IFN expression through an RNase L-MDA5 pathway.