Many potential targets of human immunodeficiency virus type 1 (HIV-1) reside in the human reproductive tract, including dendritic cells (DC). The ability of these cells to replicate HIV-1 is dependent on many factors such as their differentiation/maturation stage. Nevertheless, precise mechanisms underlying the early steps of transmucosal infection are still unknown. Our purpose was to investigate DC/HIV-1 interactions in a subepithelial mucosal layer equivalent (SEMLE) reconstructed in vitro. We used mixed interstitial DC (IntDC)/Langerhans cell (LC)-like cell subpopulations generated in vitro from CD34(+) progenitors. These cells were either integrated in SEMLE or maintained in suspension. Experimental infections were performed with a type X4 strain (HIV-1(LAI)) and a type R5 strain (HIV-1(Ba-L)). Proviral DNA was detected by in situ polymerase chain reaction (PCR) and viral replication was quantified by measuring p24 core protein release in the culture media. Our results showed that SEMLE enable DC to retain immature stage and reproduce the tropic selection that occurs in vivo. Indeed, IntDC/LC were infected by both types of HIV-1 strains, regardless of the infection schedule, whereas only type R5 virus replicated in DC in the absence of T cell subsets. Furthermore, the ability of DC to replicate HIV-1(BaL) was lost after 14 days of culture unless the cells had previously been integrated in SEMLE. These results suggest that this 3D model maintains the ability of DC to replicate type R5 virus by delaying their maturation. In conclusion, this in vitro model mimics human submucosa and can be considered as relevant for studying the preliminary steps of transmucosal HIV-1 infection.