The protein high-mobility group box 1 (HMGB1) is released into the extracellular space in response to many inflammatory stimuli, where it is a potent signaling molecule. Although research has focused on downstream HMGB1 signaling, the means by which HMGB1 exits the cell is controversial. Here we demonstrate that HMGB1 is not released from bone marrow-derived macrophages (BMDM) after lipopolysaccharide (LPS) treatment. We also explore whether HMGB1 is released via the pore-forming protein gasdermin D after inflammasome activation, as is the case for IL-1β. HMGB1 is only released under conditions that cause cell lysis (pyroptosis). When pyroptosis is prevented, HMGB1 is not released, despite inflammasome activation and IL-1β secretion. During endotoxemia, gasdermin D knockout mice secrete HMGB1 normally, yet secretion of IL-1β is completely blocked. Together, these data demonstrate that in vitro HMGB1 release after inflammasome activation occurs after cellular rupture, which is probably inflammasome-independent in vivo.