The mechanism of viral transmission across the mucosal barrier is poorly understood. Using the endometrial epithelium-derived cell line HEC-1A, we found that the cells are capable of sequestering large numbers of HIV-1 particles but are refractory to cell-free viral infection. The removal of heparan sulfate moieties of cell-surface proteoglycans (HSPG) from the apical pole of HEC-1A accounted for at least 60% of both R5- and X4-HIV-1 attachment, showing their important implication in viral attachment. HEC-1A cells also have the capacity to endocytose a weak proportion of the attached virus and pass it along to underlying cells. Fucose, N-acetylglucosamine and mannosylated-residues inhibited the transcytosis of some virus isolates, suggesting that mannose receptors can be implicated on the both R5- and X4-HIV-1 transcytosis. The inhibition of HIV transcytosis by blocking CCR5 mAb suggests the implication of specific interaction between the viral gp120 and sulfated moiety of syndecans during the transcytosis of mostly R5- and X4-HIV-1. At the basolateral pole of HEC-1A, HSPG sequestered X4- and not R5-HIV-1, highlighting the important role of HEC-1A as an X4 virus reservoir. The cell-free virus particles that have transcytosed could infect activated T cells but with a weaker efficiency than virus that had not transcytosed. The specific stimulation of HEC-1A by R5-HIV-1 increased the release of monocytes/chemokines-attracting chemokines (IL-8 and GR0) and proinflammatory cytokines (TNF-beta and IL-1alpha) that enhanced the production of virus by activated T cells. This study suggests that R5 and X4 viruses can differentially use epithelial cells to ensure their own spread.