Osteopontin is a multifunctional glycoprotein with roles in immunomodulation, inflammatory response, tissue mineralization, and tissue remodeling, which are mediated primarily through integrins. Transglutaminase 2 selectively cross-links proteins by isopeptide bonding. Osteopontin is one of the substrates of this enzyme and undergoes polymerization; however, the biological meaning of this polymerization remains unknown. Using recombinant osteopontin polymerized with purified transglutaminase 2, we examined cell adhesion, spreading, focal contact formation, and migration of SW480 or HUVE cells. All of these cellular behaviors were dramatically enhanced with polymeric osteopontin. These enhancements of cellular functions imply that polymerization might modulate physiological and pathological functions of osteopontin.