Polysialic acid (PSA) is a developmentally regulated glycan made of repeating sialic acid monomers with α2-8 linkages. PSA has very limited expression in adults, and modifies only a few cell-surface proteins. However, PSA is overexpressed in several human cancers and is associated with metastasis and poor prognosis. We have described a derivative of PSA containing a mixture of de-N-acetyl and N-acetyl neuraminic acid residues (dPSA) found intracellularly in many normal human tissues but expressed at much higher levels on the cell surface of many human cancer cell lines. The proteins modified with dPSA and dPSA function in normal and abnormal human biology are unknown. The purpose of this study was to identify protein(s) modified with PSA and possible dPSA-dependent functions in cancer cell lines that express dPSA antigens. Using co-immunoprecipitation with the anti-dPSA monoclonal antibody SEAM 2 and mass spectroscopy, we identified membrane-associated nucleolin that is either directly modified or associated with dPSA. In addition, knocking down expression of the polysialyltransferase ST8SiaII (STX) in SK-MEL-28 human melanoma cells nearly eliminated dPSA and nucleolin from membranes but had no effect on the levels of nuclear nucleolin, and resulted in aberrant cell morphology, cell adhesion, and motility. The data suggest that cell-surface nucleolin depends on modification with dPSA, and that dPSA-modified nucleolin has an important role in cell adhesion and migration.