The study investigates the role of high mobility group box 1 (HMGB1) in regulating autophagy. HMGB1, a chromatin-associated nuclear protein and extracellular damage-associated molecular pattern molecule, is shown to be a critical regulator of autophagy. Stimuli that enhance reactive oxygen species (ROS) promote cytosolic translocation of HMGB1, which in turn enhances autophagic flux. HMGB1 directly interacts with the autophagy protein Beclin 1, displacing Bcl-2. Depletion of HMGB1 inhibits autophagy and increases apoptosis, suggesting that HMGB1 is essential for maintaining autophagy and limiting apoptosis. The oxidation of HMGB1 regulates its subcellular localization and autophagic function, with the C106 residue being crucial for nuclear localization and the C23/C45 disulfide bridge required for binding to Beclin 1. These findings highlight a novel role for HMGB1 in promoting cell survival by sustaining autophagy under cellular stress conditions.The study investigates the role of high mobility group box 1 (HMGB1) in regulating autophagy. HMGB1, a chromatin-associated nuclear protein and extracellular damage-associated molecular pattern molecule, is shown to be a critical regulator of autophagy. Stimuli that enhance reactive oxygen species (ROS) promote cytosolic translocation of HMGB1, which in turn enhances autophagic flux. HMGB1 directly interacts with the autophagy protein Beclin 1, displacing Bcl-2. Depletion of HMGB1 inhibits autophagy and increases apoptosis, suggesting that HMGB1 is essential for maintaining autophagy and limiting apoptosis. The oxidation of HMGB1 regulates its subcellular localization and autophagic function, with the C106 residue being crucial for nuclear localization and the C23/C45 disulfide bridge required for binding to Beclin 1. These findings highlight a novel role for HMGB1 in promoting cell survival by sustaining autophagy under cellular stress conditions.