Respiratory syncytial virus (RSV) is the leading cause of respiratory viral infection in infants and children, yet little is known about the antiviral response of plasmacytoid dendritic cells (pDCs) to RSV infection. We tracked the cellular source of interferon-beta using interferon-beta/yellow fluorescent protein (YFP) reporter mice and identified the signaling pathway activated by RSV that induces type I interferon production in pDCs and DCs. Results from in vitro analyses of RSV-stimulated bone marrow cells revealed that RSV induces interferon-beta production in both pDCs and DCs. Kinetic analyses of interferon-beta-producing cells in RSV-infected lung cells in vivo indicated that pDCs are rapidly recruited to sites of inflammation during infection. These cells produced interferon-beta via the TLR7-MyD88-mediated pathway and IFN alpha 1R-mediated pathway rather than the MAVS-mediated pathway. Moreover, pDC-ablated mice exhibited decreased interferon-gamma production and the antigen specificity of CD8(+) T cells. Collectively, these data indicate that pDCs play pivotal roles in cytotoxic T lymphocyte (CTL) responses and are one of producers of type I interferon during RSV infection.