High mobility group box 1 (HMGB1) is a highly conserved protein with multiple intracellular and extracellular functions, including transcriptional regulation, as well as modulation of inflammation, cell migration, and ingestion of apoptotic cells. In these experiments, we examined a potential role for intracellular HMGB1 in modulating phagocytosis.We found that phagocytosis of apoptotic cells resulted in translocation of HMGB1 into the cytoplasm and extracellular space. Transient or stable inhibition of HMGB1 expression in bone marrow-derived macrophages or fibroblasts resulted in increased phagocytosis of apoptotic thymocytes and apoptotic neutrophils. Knockdown of HMGB1 was associated with enhanced activation of Rac-1 and cytoskeletal rearrangement. Intracellular events involved in phagocytosis and upstream of Rac-1 activation, such as phosphorylation of ERK and focal adhesion kinase (FAK), were increased after knockdown of HMGB1. Inhibition of Src kinase activity prevented the increase in phosphorylation of FAK and ERK present during phagocytosis in HMGB1 knockdown cells, and also abrogated the enhancement in phagocytosis associated with HMGB1 knockdown. Interaction between Src and FAK in the cytoplasm of HMGB1 knockdown fibroblasts was enhanced compared with that present in control fibroblasts. Under in vitro conditions, the presence of HMGB1 diminished interactions between purified FAK and Src. These studies demonstrate a novel role for HMGB1 in the regulation of phagocytosis. In particular, these experiments show that intracellular HMGB1, through associating with Src kinase and inhibiting interactions between Src and FAK, diminishes the phagocytic ability of macrophages and other cell populations. The Journal of Immunology, 2011, 187: 4686-4694. Copyright © 2011 by The American Association of Immunologists, Inc.