DC-SIGN, a C-type lectin mainly expressed by DCs, mediates antigen uptake and can induce specific immune responses, depending on the ligand involved. Owing to these properties, DC-SIGN is an attracting target for approaches aimed at tailoring the immune response towards specific immunologic outcomes. A multivalent DC-SIGN ligand (Polyman26), containing at its core a fluorescent "rod-like" spacer and able to inhibit DC-SIGN mediated HIV infection in nanomolar concentration, has been recently developed by our group. We investigated the internalization pattern and the ability of Polyman26 to elicit innate immune responses. Results obtained by confocal microscopy indicate that Polyman26 is internalized by DCs via receptor- mediated endocytosis and is then routed to endolysosomal compartments, thus being presented together with MHC class II molecules, with important implications for the development of vaccines. Moreover, Polyman26 up-regulated the production of β-chemokines and pro-inflammatory cytokines (including IL-1β, IL-6, IL-12, and TNFα) as well as the expression of TLR9 and CD40L. These results indicate that glycomimetic DC-SIGN ligands should be further investigated and suggest that these compounds could be used to differentially stimulate immune responses.