Type I IFNs play an important role in the immune response to enterovirus infections. Their importance is underscored by observations showing that many enteroviruses including coxsackie B viruses (CVBs) have developed strategies to block type I IFN production. Recent studies have highlighted a role for the type III IFNs (also called IFNλs) in reducing permissiveness to infections with enteric viruses including coxsackievirus. However, whether or not CVBs have measures to evade the effects of type III IFNs remains unknown. By combining virus infection studies and different modes of administrating the dsRNA mimic poly I : C, we discovered that CVBs target both TLR3- and MDA5/RIG-I-mediated type III IFN expression. Consistent with this, the cellular protein expression levels of the signal transduction proteins TRIF and IPS1 were reduced and no hyperphosphorylation of IRF-3 was observed following infection with the virus. Notably, decreased expression of full-length TRIF and IPS1 and the appearance of cleavage products was observed upon both CVB3 infection and in cellular protein extracts incubated with recombinant 2Apro, indicating an important role for the viral protease in subverting the cellular immune system. Collectively, our study reveals that CVBs block the expression of type III IFNs, and that this is achieved by a similar mechanism as the virus uses to block type I IFN production. We also demonstrate that the virus blocks several intracellular viral recognition pathways of importance for both type I and III IFN production. The simultaneous targeting of numerous arms of the host immune response may be required for successful viral replication and dissemination.