The human scavenger receptor gp340 has been identified as a binding protein for the human immunodeficiency virus type 1 (HIV-1) envelope that is expressed on the cell surface of female genital tract epithelial cells. This interaction allows such epithelial cells to efficiently transmit infective virus to susceptible targets and maintain viral infectivity for several days. Within the context of vaginal transmission, HIV must first traverse a normally protective mucosa containing a cell barrier to reach the underlying T cells and dendritic cells, which propagate and spread the infection. The mechanism by which HIV-1 can bypass an otherwise healthy cellular barrier remains an important area of study. Here, we demonstrate that genital tract-derived cell lines and primary human endocervical tissue can support direct transcytosis of cell-free virus from the apical to basolateral surfaces. Further, this transport of virus can be blocked through the addition of antibodies or peptides that directly block the interaction of gp340 with the HIV-1 envelope, if added prior to viral pulsing on the apical side of the cell or tissue barrier. Our data support a role for the previously described heparan sulfate moieties in mediating this transcytosis but add gp340 as an important facilitator of HIV-1 transcytosis across genital tract tissue. This study demonstrates that HIV-1 actively traverses the protective barriers of the human genital tract and presents a second mechanism whereby gp340 can promote heterosexual transmission.