Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to blood transfusion and sexual transmission, HTLV-1 is transmitted mainly through prolonged breastfeeding, and such infection represents a major risk for the development of adult T-cell leukemia/lymphoma. Although HTLV-1-infected lymphocytes can be retrieved from maternal milk, the mechanisms of HTLV-1 transmission through the digestive tract remain unknown. In the present study, we assessed HTLV-1 transport across the epithelial barrier using an in vitro model. Our results show that the integrity of the epithelial barrier was maintained during coculture with HTLV-1-infected lymphocytes, because neither morphological nor functional alterations of the cell monolayer were observed. Enterocytes were not susceptible to HTLV-1 infection, but free infectious HTLV-1 virions could cross the epithelial barrier via a transcytosis mechanism. Such virions were able to infect productively human dendritic cells located beneath the epithelial barrier. Our data indicate that HTLV-1 crosses the tight epithelial barrier without disruption or infection of the epithelium to further infect target cells such as dendritic cells. The present study provides the first data pertaining to the mode of HTLV-1 transport across a tight epithelial barrier, as can occur during mother-to-child HTLV-1 transmission during breastfeeding.