Dendritic cells (DCs) are an attractive tool for immunomodulation, targeting mature DCs (mDCs) for immunization or immature/semimature DCs (iDCs) for tolerization. Therefore, introducing antigens into DCs has become a prime topic in various immunological disciplines. Numerous studies have shown that lentiviruses are an efficient vehicle for this purpose. This study evaluates the effects of lentiviral transduction on iDC activation. Immature DCs are efficiently transduced with increasing doses of lentivirus without affecting cell viability. Transduction at low multiplicities of infection (MOIs) did not result in phenotypical or functional maturation. Higher doses of lentivirus, however, resulted in upregulation of adhesion, costimulatory, and HLA molecules, as well as in increased allostimulatory capacity and secretion of interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha. Production of IL-12 p70, IL-10, and interferon-alpha was observed only at extremely high doses. Protein kinase R phosphorylation on transduction at an MOI of 150 was demonstrated by Western blotting. A Toll-like receptor (TLR)-driven luciferase reporter assay showed dose-dependent activation of TLR2, TLR3, and TLR8, which was independent of the pseudotype, production, or transduction protocol and was abrogated on heat inactivation. These data show that lentiviral vectors provide not only the antigen but also appropriate activation signals to iDCs, favoring their use for immunotherapy and vaccine development.