Plasmacytoid dendritic cells (pDCs) play a critical role in antiviral immunity through their ability to produce large amounts of type I IFNs. Activation of pDCs upon viral infection has been shown to be dependent on MyD88 and mediated by Toll-like receptors (TLR) 7 and 9, which sense viral ssRNA and CpG DNA, respectively. In this study, we showed that murine pDC recognition of vaccinia virus (VV), a dsDNA virus, was MyD88-dependent but TLR9-independent. Using HEK293 cells transfected with murine TLR7 or TLR8 and a NF-kappaB luciferase reporter, we demonstrated that stimulation of TLR8-, but not TLR7-, transfected cells with either VV or VV DNA resulted in substantial NF-kappaB activation, and that siRNA-mediated knockdown of TLR8 expression in pDCs led to a complete ablation of VV-induced type I IFN production. We further identified that the VV genome was rich in poly(A)/T sequences, and synthetic poly(A) and poly T oligodeoxynucleotides were capable of activating pDCs in a TLR8-dependent manner. In vivo, TLR8-MyD88-dependent pDC activation played a critical role in innate immune control of VV infection. Collectively, our data are unique in demonstrating that TLR8 is required for sensing poly(A)/T-rich DNA in pDCs, and that murine TLR8 is functional in the context of a viral infection.