High-mobility group box 1 (HMGB1) protein is a member of the highly conserved non-histone DNA binding protein family. First identified in 1973, as one of a group of chromatin-associated proteins with high acidic and basic amino acid content, it was so named for its characteristic rapid mobility in polyacrylamide gel electrophoresis. HMGB1 was later discovered to have another function. It is released from a variety of cells into the extracellular milieu to act on specific cell-surface receptors. In this latter role, HMGB1 is a proinflammatory cytokine that may contribute to many inflammatory diseases, including sepsis. Therefore, HMGB1 regulates intracellular cascades influencing immune cell functions, including chemotaxis and immune modulation. The bioactivity of the HMGB1 is determined by specific posttranslational modifications that regulate its role in inflammation and immunity. During tumor development, HMGB1 has been reported to play paradoxical roles in promoting both cell survival and death by regulating multiple signaling pathways. In this review, we focus on the role of HMGB1 in physiological and pathological responses, as well as the mechanisms by which it contributes to immunity, inflammation, and cancer progression.