The multifunctional protein nucleolin (NCL) is overexpressed on the surface of activated endothelial and tumor cells and mediates the stimulatory actions of several angiogenic growth factors, such as pleiotrophin (PTN). Because α(v)β(3) integrin is also required for PTN-induced cell migration, the aim of the present work was to study the interplay between NCL and α(v)β(3) by using biochemical, immunofluorescence, and proximity ligation assays in cells with genetically altered expression of the studied molecules. Interestingly, cell surface NCL localization was detected only in cells expressing α(v)β(3) and depended on the phosphorylation of β(3) at Tyr(773) through receptor protein-tyrosine phosphatase β/ζ (RPTPβ/ζ) and c-Src activation. Downstream of α(v)β(3,) PI3K activity mediated this phenomenon and cell surface NCL was found to interact with both α(v)β(3) and RPTPβ/ζ. Positive correlation of cell surface NCL and α(v)β(3) expression was also observed in human glioblastoma tissue arrays, and inhibition of cell migration by cell surface NCL antagonists was observed only in cells expressing α(v)β(3). Collectively, these data suggest that both expression and β(3) integrin phosphorylation at Tyr(773) determine the cell surface localization of NCL downstream of the RPTPβ/ζ/c-Src signaling cascade and can be used as a biomarker for the use of cell surface NCL antagonists as anticancer agents.