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 α(v)β(3) as well as α(v)β(5)-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 α(v)β(3), but not with soluble α(v)β(5). HMGB1 colocalized with the β(3)-integrin on the cell membrane of macrophages and bound to soluble α(v)β(3), but not to soluble α(v)β(5). HMGB1 suppressed the interaction between MFG-E8 and α(v)β(3). 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 α(v)β(3)-dependent recognition and uptake of apoptotic cells.