The degree and duration of immunity against herpes simplex virus type 2 (HSV-2) infection of the female genital tract were assessed after intranasal (i.nl.) or intraperitoneal (i.p.) immunization with a recombinant adenovirus vector expressing HSV glycoprotein B (AdgB8). After intravaginal HSV-2 challenge, control mice rapidly developed disease and displayed high virus titers in vaginal washes. In contrast, virus titers decreased significantly and at similar rates in i.nl. and i.p. immunized mice and by day 7 were undetectable in vaginal wash samples. Assessment of genital pathology and survival showed that only i.nl. immunization provided long-term protection. Examination of antibody-secreting cells (ASCs) during the decline in vaginal virus titers revealed that gB-specific IgA ASCs were only observed in the genital tissues of i.nl. immunized mice. These results indicate that mucosal immunization provides a high and long-lasting level of immunity from sexually transmitted viral infections of the female genital tract.