Immature dendritic cells (iDCs) are likely to be among the first targets of HIV infection during sexual transmission. We analyzed whether the relatively inefficient viral replication in iDCs could be attributed to specific restrictions during the viral life cycle. Using iDCs from a panel of donors, we set out to compare their capacity to support infection and propagation of X4- and R5-tropic viruses. We also performed quantitative flow cytometry to determine levels of relevant cell-surface CD4 and HIV-1 co-receptors. Although iDCs express comparable levels of functional CXC chemokine receptor 4 (CXCR4) and CC chemokine receptor 5 (CCR5) at the cell surface, they are 100- to 1,000-fold less susceptible to infection by X4- versus R5-tropic HIV-1 strains. Increasing surface expression of CXCR4 by transduction with lentiviral vectors did not lead to increased replication of the X4-tropic strains. Fusion of HIV-X4 with iDCs was markedly less efficient compared to that of HIV-R5. We conclude that an env-specific block early in the viral cycle operates in iDCs. This restriction may play a role in the exclusion of X4-tropic strains during HIV-1 transmission.