SummaryThe polysialic acid (polySia) modification of the neural cell adhesion molecule NCAM is a key regulator of cell migration. Yet its role in NCAM-dependent or NCAM-independent modulation of motility and cell-matrix adhesion is largely unresolved. Here, we demonstrate that loss of polySia attenuates tumour cell migration and augments the number of focal adhesions in a cell-cell contact-and NCAM-dependent manner. In the presence or absence of polySia, NCAM never colocalised with focal adhesions but was enriched at cell-cell contacts. Focal adhesion of polySia-and NCAM-negative cells was enhanced by incubation with soluble NCAM or by removing polySia from heterotypic contacts with polySia-NCAM-positive cells. Focal adhesion was compromised by the src-family kinase inhibitor PP2, whereas loss of polySia or exposure to NCAM promoted the association of p59Fyn with the focal adhesion scaffolding protein paxillin. Unlike other NCAM responses, NCAM-induced focal adhesion was not prevented by inhibiting FGF receptor activity and could be evoked by NCAM fragments comprising immunoglobulin domains three and four but not by the NCAM fibronectin domains alone or by an NCAM-derived peptide known to interact with and activate FGF receptors. Together, these data indicate that polySia regulates cell motility through NCAM-induced but FGF-receptor-independent signalling to focal adhesions.