The present study was undertaken to examine the potential of rectal route of immunization for induction of protective immunity in the female genital tract against genital herpes infection in mice. A single rectal immunization of female C57Bl/6 mice with live attenuated herpes simplex virus type 2 lacking thymidine kinase (HSV-2 TK-) was shown to confer HSV-specific cellular and humoral immune responses as well as protection against an otherwise lethal vaginal challenge with a virulent HSV-2 strain. The immunity afforded by rectal immunization with HSV-2 TK- was shown to be independent of sex hormonal influence and the usage of the adaptor protein myeloid differentiation factor 88 (MyD88). Next, the impact of rectal immunization with HSV-2 glycoprotein D (gD) in combination with CpG oligodeoxynucleotide (ODN) or cholera toxin (CT) on induction of immunity against HSV-2 was investigated. Rectal immunization of mice with gD+CpG failed to generate gD specific immune responses and protection against genital herpes infection. Conversely, rectal immunization with gD+CT elicited potent gD-specific cellular immune responses and protection against genital herpes infection through a MyD88-dependent manner. These results highlight the potential of rectal route for the development of novel immunization strategies to elicit immunity in the female genital tract against genital herpes and presumably other sexually transmitted diseases.