Herpes simplex virus (HSV)-1 has been discovered in placental tissue from spontaneous miscarriages, but reports of transplacental transmission and fetal infection are extremely rare. Previously, we demonstrated that the villous syncytiotrophoblast, which forms a continuous layer between the maternal and fetal circulation, is resistant to HSV entry. Here, we tested our hypothesis that the villous syncytiotrophoblast prevents transplacental transmission of HSV secondary to decreased expression of HSV entry mediators (HveA, HveB, and HveC). In addition, we investigated the ability of HSV to infect extravillous trophoblast cells, which mediate placental attachment to the uterine wall, and the expression of HSV receptors in these cells. We performed fluorescence-activated cell sorting (FACS) analyses and immunostaining to demonstrate that HveA, HveB, and HveC were not expressed in third-trimester villous trophoblast cells. Consequently, villous explants obtained from third-trimester placentas were resistant to infection by a recombinant HSV-1 vector, HSV-1 KOS, but approximately 20% of mesenchymal cells within the villous core were infected when villous explants were pretreated with trypsin to disrupt the villous trophoblast layer. Conversely, FACS analysis and immunostaining demonstrated that extravillous trophoblast cells expressed HveA, HveB, and HveC, and these cells were efficiently infected by HSV vectors. Infection of extravillous trophoblast cells by HSV-1 was not reduced when the cells were pretreated with an antibody against HveA but was partially reduced when the cells were pretreated with antibodies directed against HveB and HveC. Thus, the decreased expression of herpesvirus entry mediators in villous syncytiotrophoblast prevents placental villous infection, thereby limiting maternal-fetal transmission of HSV.