Osteopontin (OPN) is highly expressed by macrophages and plays a key role in the pathology of several chronic inflammatory diseases including atherosclerosis and the foreign body reaction. However, the molecular mechanism behind OPN regulation of macrophage functions is not well understood. OPN is a secreted molecule and interacts with several integrins via two domains: the RGD sequence binding to αv-containing integrins, and the SLAYGLR sequence binding to α4β1, α4β7, and α9β1 integrins. Here we determined the role of OPN in macrophage survival, chemotaxis, and activation state. For survival studies, OPN treated-bone marrow derived macrophages (BMDMs) were challenged with growth factor withdrawal and neutralizing integrin antibodies. We found that survival in BMDMs is mediated primarily through the α4 integrin. In chemotaxis studies, we observed that migration to OPN was blocked by neutralizing α4 and α9 integrin antibodies. Further, OPN did not affect macrophage activation as measured by IL-12 production. Finally, the relative contributions of the RGD and the SLAYGLR functional domains of OPN to leukocyte recruitment were evaluated in an in vivo model. We generated chimeric mice expressing mutated forms of OPN in myeloid-derived leukocytes, and found that the SLAYGLR functional domain of OPN, but not the RGD, mediates macrophage accumulation in response to thioglycollate-elicited peritonitis. Collectively, these data indicate that α4 and α9 integrins interacting with OPN via the SLAYGLR domain play a key role in macrophage biology by regulating migration, survival, and accumulation. J. Cell. Biochem. 114: 1194–1202, 2013. © 2012 Wiley Periodicals, Inc.