Dendritic cells (DCs) need to mobilize within the extracellular matrix (ECM) during their maturation and concomitant migration from peripheral sites to lymphoid organs. Syndecans are cell surface proteoglycans that mediate the interaction of DCs with the ECM. Here we investigated the influence of syndecans on dendritic cell motility and morphology. Langerhans cells of the epidermis and monocyte-derived DCs were found to undergo a switch in syndecan expression during maturation. Syndecan-1 was downregulated and syndecan-4 was strongly upregulated within the first hours of lipopolysaccharide-induced dendritic cell maturation and during Langerhans cell emigration from human skin, as shown by flow cytometry and qRT-PCR. Syndecan-1 downregulation was inhibited by syndecan-4 siRNA knock-down, indicating a functional interconnection between enhanced syndecan-4 expression and syndecan-1 downregulation. Syndecan-4 upregulation is functionally involved in dendritic cell motility, as inhibition of syndecan-4 function by means of blocking antibodies or through siRNA knock-down decreased dendritic cell motility. In other experiments, the cytoskeletal component a-actinin was observed to be upregulated in DCs as a consequence of the induction of maturation, and was found to colocalize with syndecan-4. Furthermore, lammellopodial spreading by DCs on fibronectin (FN)-coated surfaces was dependent on syndecan-4. This binding of syndecan-4 to FN and its association with the cytoskeleton may be relevant for syndecan-4-dependent dendritic cell motility. We conclude that the switch in syndecan expression during dendritic cell maturation controls the motility of DCs in a way that appears to be crucial for their mobilization from peripheral sites and subsequent migration to lymphoid tissues.