Dendritic cells (DCs) are powerful APCs capable of activating naive lymphocytes. Of the DC subfamilies, plasmacytoid DCs (pDCs) are unique in that they secrete high levels of type I IFNs in response to viruses but their role in inducing adaptive immunity remains divisive. In this study, we examined the importance of pDCs and their ability to recognize a virus through TLR9 in immunity against genital HSV-2 infection. We show that a low number of pDCs survey the vaginal mucosa at steady state. Upon infection, pDCs are recruited to the vagina and produce large amounts of type I IFNs in a TLR9-dependent manner and suppress local viral replication. Although pDCs are critical in innate defense against genital herpes challenge, adaptive Th1 immunity developed normally in the absence of pDCs. Thus, by way of migrating directly into the peripheral mucosa, pDCs act strictly as innate antiviral effector cells against mucosal viral infection in situ.