Genital epithelial cells (ECs) are the first line of defense that sexually transmitted viruses encounter. The mechanism of viral pathogenesis in these cells is not well understood. Here, we show that a primary cell culture model from human reproductive tract tissues can be used as a novel ex vivo model in examining the interaction of herpes simplex virus, type 2 (HSV-2), with female genital mucosa. Confluent, polarized primary cultures of human endometrial and cervical ECs were established and shown to be free from any significant contamination of any other cell type. Both endometrial and cervical ECs were found to be highly susceptible to HSV-2 infection. The kinetic of infection was similar to in vivo infection, with the earliest viral shedding seen at 18 h postinfection. Primary EC monolayers could be infected both apically and basolaterally, but preferential viral shedding was seen on the apical side of cells. Following treatment of the monolayers with poly (I:C), an innate immune activator that acts via TLR3, viral shedding was reduced significantly, comparable to levels seen when an antiviral formulation, acyclovir, was used. Treatment of epithelial and stromal co-cultures with estradiol increased HSV-2 infection in endometrial ECs, but viral shedding decreased following treatment with progesterone. To the best of our knowledge, this is the first study that examines the interaction of primary human female genital ECs with HSV-2, using an ex vivo culture model. The results provide valuable information regarding the susceptibility of women's genital ECs to HSV-2 and the ability of innate immunity and hormones to modify this susceptibility.