Osteoclasts from osteopontin-efficient mice exhibit decreased CD44 surface expression. Osteopontin (OPN)/αvβ3 generated Rho signaling pathway is required for the surface expression of CD44. In this work we show the Rho effector, Rho kinase (ROK-α), to be a potent activator of CD44 surface expression. ROK-α activation was associated with autophosphorylation, leading to its translocation to the plasma membrane, as well as its association with CD44. ROK-α promoted CD44 surface expression through phosphorylation of CD44 and ezrin-radixin-moesin (ERM) proteins and CD44·ERM·actin complex formation. Osteoclasts from OPN-/- mice exhibited an ∼55-60% decrease in basal level ROK-α phosphorylation as compared with wild type osteoclasts. Furthermore, RhoVal-14 transduction was only partially effective in stimulating ROK-α/CD44 phosphorylation, as well as CD44 surface expression, in these osteoclasts. Studies on the inhibition of Rho by C3 transferase or ROK-α by the specific inhibitor, Y-27632, showed a decrease in the phosphorylation mediated by ROK-α and CD44 surface expression. Neutralizing antibodies to αvβ3- or CD44 inhibited the migration and bone resorption of wild type osteoclasts. However, only anti-αv or -β3, antibodies blocked OPN-induced phosphorylation of ROK-α, CD44, and the ERM proteins. Our results strongly suggest a role for ROK-α in αvβ3-mediated Rho signaling, which is required for the phosphorylation events and CD44 surface expression. The functional deficiencies in the Rho effector(s) because of the lack of OPN were associated with decreased CD44 surface expression and hypomotility in the OPN-/- osteoclasts. Finally, we find that cooperativity exists between αvβ3 and CD44 for osteoclast motility and bone resorption.