The purpose of this study was to evaluate the kinetic changes and the localization of high-mobility group box 1 protein (HMGB1) and to observe the effect of heat shock response (HSR) on the expression and release of HMGB1 in lipopolysaccharide (LPS)-activated murine macrophage-like RAW 264.7 cells. Reverse transcriptase (RT)-PCR and Western blot were used to examine HMGB1 expression after LPS treatment. The intracellular localization of HMGB1 in normal or LPS-activated cells was investigated by immunocytochemical analysis and HMGB1 released from cultured macrophages by Western blot. HSR was performed by incubating RAW 264.7 cells at 42.5°C for 1 h then recovery at 37°C for 12 h. The effect of HSR on expression and release of HMGB1 was observed. The results showed that a decrease of HMGB1 mRNA expression was observed at 18 h after LPS (500 ng/mL) treatment, although the total intracellular HMGB1 protein levels were not affected. A visible translocation of HMGB1 from the nuclear to the cytoplasm was observed at 20 h after stimulation with LPS (500 ng/mL). Furthermore, HMGB1 was released into the medium by LPS-activated RAW 264.7 cells in a time- and dose-dependent manner. Heat shock pretreatment significantly inhibited LPS-induced release of HMGB1 and the translocation of HMGB1 from the nucleus to the cytoplasm in RAW 264.7 cells. These findings suggest that the release of HMGB1 by LPS-activated macrophages is a late event in the pathogenesis of sepsis and that HSR could inhibit the release and translocation of HMGB1 induced by LPS.