Sexual transmission is now the most frequent means of diffusion of human immunodeficiency virus type 1 (HIV-1). Even if the underlying mechanism is still largely unknown, there is a consensus regarding the key role played by mucosal dendritic cells (DCs) in capturing HIV through contact with infected subepithelial lymphocytes, and their capacity to spread HIV by trans-infection. We found that HIV protease inhibitors (PIs) reduced virion endocytosis strongly in monocyte-derived immature (i) DCs contacting HIV-1-infected cells, and that this phenomenon led to dramatically impaired trans-infection activity. This inhibitory effect was not mediated by the block of viral protease activity, as it was also operative when donor cells were infected with a PI-resistant HIV-1 strain. The block of virus maturation imposed by PIs did not correlate with significant variations in the levels of virus expression in donor cells or of Gag/Env virion incorporation. Also, PIs did not affect the endocytosis activity of DCs. In contrast, we noticed that PI treatment inhibited the formation of cell-cell conjugates whilst reducing the expression of ICAM-1 in target iDCs. Our results contribute to a better delineation of the mechanisms underlying HIV-1 trans-infection activity in DCs, whilst having implications for the development of new anti-HIV microbicide strategies.