Syndecans are cell‐surface heparan sulfate proteoglycans, which perform a variety of functions in the cell. Most important, they are co‐receptors for growth factors and mediate cell–cell and cell–matrix interactions. Four syndecans (syndecan 1–4) have been described in different species. The aim of this work was the cloning and characterization of human syndecan‐3. The human syndecan‐3 sequence has high homology to the rat and mouse sequences, with the exception of the 5′‐region. Syndecan‐3 mRNA is mostly expressed in the nervous system, the adrenal gland, and the spleen. When different cell lines were transiently transfected with full‐length syndecan‐3 cDNA, it was localized to the membrane and induced the formation of long filopodia‐like structures, microspikes, and varicosities. Consequently, the actin cytoskeleton was re‐organized, since actin staining was mostly found in the cellular extensions and at the cell periphery, co‐localizing with the syndecan‐3 staining. The development of the phenotype depended on the presence of sugar chains, as transfected glycosaminoglycan‐deficient Chinese hamster ovary (CHO) 745 cells did not show these structural changes, nor did transfected CHO K1 cells in the presence of heparin. The similarity of the cloned DNA sequence with that of other mammalian species and the high expression in the nervous system led us to the assumption that human syndecan‐3 could perform comparable functions to those described for syndecan‐3 in rat and mouse. Additionally, transient transfection experiments suggest a role of human syndecan‐3 in the organization of cell shape by affecting the actin cytoskeleton, possibly by transferring signals from the cell surface in a sugar‐dependent mechanism. J. Cell. Biochem. 82: 246–259, 2001. © 2001 Wiley‐Liss, Inc.