Osteopontin (OPN) is a multifunctional phosphorylated protein containing the integrin binding sequence Arg-Gly-Asp through which it interacts with several integrin receptors, such as the α(V)β(3)-integrin. OPN exists in many different isoforms differing in phosphorylation status that are likely to interact differently with integrins. The C-terminal region of OPN is particularly well conserved among mammalian species, which suggests an important functional role of this region. In this study, we show that modification of the extreme C terminus of OPN plays an important regulatory role for the interaction with the α(V)β(3)-integrin. It is demonstrated that highly phosphorylated OPN has a much reduced capability to promote cell adhesion via the α(V)β(3)-integrin compared with lesser phosphorylated forms. The cell attachment promoted by highly phosphorylated OPN could be greatly increased by both dephosphorylation and proteolytic removal of the C terminus. Using recombinantly expressed OPN containing a tag in the N or C terminus, it is shown that a modification in the C-terminal part significantly reduces the adhesion of cells to OPN via the α(V)β(3)-integrin, whereas modification of the N terminus does not influence the binding. The inhibited binding of the α(V)β(3)-integrin to OPN could be restored by proteolytic removal of the C terminus by thrombin and plasmin. These data illustrate a novel mechanism regulating the interaction of OPN and the α(V)β(3)-integrin by modification of the highly conserved C-terminal region of the protein.