Phagocytosis of apoptotic cells is critical to resolution of inflammation. High mobility group box 1 protein (HMGB1), a mediator of inflammation, has been shown to diminish phagocytosis through binding to phosphatidylserine (PS) exposed on the surface of apoptotic neutrophils. However, it is currently unknown whether HMGB1 also modulates the activity of receptors involved in PS recognition on the surface of phagocytes. In the present studies, we found that preincubation of macrophages with HMGB1 decreased their ability to engulf apoptotic neutrophils or thymocytes. Preincubation of macrophages with HMGB1 prevented the enhancement of efferocytosis resulting from exposure to milk fat globule EGF factor 8 (MFG-E8), an opsonin that bridges PS and αvβ3 as well as αvβ5-integrins on the surface of phagocytes. The inhibitory effect of HMGB1 on the phagocytic activity of macrophages was prevented by preincubation of HMGB1 with soluble αvβ3, but not with soluble αvβ5. HMGB1 colocalized with the β3-integrin on the cell membrane of macrophages and bound to soluble αvβ3, but not to soluble αvβ5. HMGB1 suppressed the interaction between MFG-E8 and αvβ3. HMGB1 also inhibited intracellular signaling events, including ERK phosphorylation and Rac-1 activation, which are activated in macrophages during phagocytosis of apoptotic cells. These results demonstrate that HMGB1 blocks αvβ3-dependent recognition and uptake of apoptotic cells.