Syndecan-4, a cell surface heparan sulfate proteoglycan, is known to regulate the organization of the cytoskeleton, and oligomerization is crucial for syndecan-4 function. We therefore explored a possible regulatory effect of syndecan-4 oligomerization on the cytoskeleton. Glutathione-S-transferase-syndecan-4 proteins were used to show that syndecan-4 interacted specifically with alpha-actinin, but not paxillin, talin, and vinculin. Interestingly, only dimeric, and not monomeric, recombinant syndecan-4 interacted with alpha-actinin in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2), and PIP2 potentiated the interaction of both the cytoplasmic domain syndecan-4 peptide and recombinant syndecan-4 proteins with alpha-actinin, implying that oligomerization of syndecan-4 was important for this interaction. Consistent with this notion, alpha-actinin interaction was largely absent in syndecan-4 mutants defective in transmembrane domain-induced oligomerization, and alpha-actinin-associated focal adhesions were decreased in rat embryo fibroblasts expressing mutant syndecan-4. Besides, this interaction was consistently lower with the phosphorylation-mimicking syndecan-4 mutant S183E which is known to destabilize the oligomerization of the syndecan-4 cytoplasmic domain. Taken together, the data suggest that the oligomeric status of syndecan-4 plays a crucial role in regulating the interaction of syndecan-4 with alpha-actinin.