Opsonization of apoptotic cells facilitates recognition by phagocytes for the rapid clearance of potentially inflammatory cellular material. The secreted glycoprotein Milk Fat Globule Factor-E8 (MFG-E8) is a member of this family of bridging molecules and is believed to bind phosphatidylserine (PS) on the dying cell, linking it to integrin receptors on the phagocyte. Here we report the characterization of a functional signaling module involving MFG-E8, alphavbeta5 integrin, and DOCK180 for the activation of Rac1. We show that MFG-E8 and DOCK180 are both expressed in phagocytic-competent primary immature dendritic cells (DCs) and DC2.4 cells, and are potently down-regulated upon DC maturation, consistent with their role in phagocytosis and antigen capture. Coexpression of MFG-E8 with alphavbeta5 integrin potentiated integrin-mediated Rac1 activation, which was abrogated by mutagenesis in the RGD motif in MFG-E8. Moreover, expression of antisense DOCK180 abrogated MFG-E8-alphavbeta5-mediated Rac activation and impaired the phagocytosis of apoptotic cells. These data demonstrate a biochemical link between an opsonin of apoptotic cells, the alphavbeta5 integrin, and the Crk-DOCK180-Rac1 pathway, and importantly, show that MFG-E8 and DOCK180 are expressed according to the functional status of the phagocyte.