Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within the context of certain flanking bases (CpG motifs) have been shown to induce potent innate and adaptive immune responses. Vaginal delivery of CpG ODN alone protects mice from vaginal herpes simplex virus type‐2 (HSV‐2) challenge. Here, we investigated the importance of timing of delivery, formulation, route and dose of vaginally administered CpG ODN in the prevention or treatment of intravaginal (IVAG) HSV‐2 infection. Mice treated intravaginally with CpG ODN containing a phosphorothioate backbone 24 hours prior to IVAG HSV‐2 challenge survived infection, showed minimal vaginal pathology, and had virtually no detectable virus in vaginal washes, when compared to mice treated with non‐CpG ODN. Genital treatment of HSV‐2 infected mice with CpG ODN 4 hours after infection resulted in increased survival and decreased pathology and vaginal virus titers, whereas treatment of infected mice with CpG ODN 24 and 72 hours after IVAG HSV‐2 infection had no effect on disease progression. Both liquid and solid (delivered on a bio‐erodible muco‐adhesive film) formulations of CpG ODN were effective in protection against genital HSV‐2 following vaginal delivery. Lastly, IVAG delivery of 10 μg of CpG ODN protected as well as a 100 μg dose. J. Med. Virol. 71:561–568, 2003. © 2003 Wiley‐Liss, Inc.