HMGB1, a highly conserved nonhistone DNA-binding protein, plays an important role in inflammatory diseases. Once released to the extracellular space, HMGB1 acts as a proinflammatory cytokine that triggers inflammatory reaction. Our previous study showed that salidroside exerts anti-inflammatory effect via inhibiting the JAK2-STAT3 signalling pathway. However, whether salidroside inhibits the release of HMGB1 is still unclear. In this study, we aim to study the effects of salidroside on HMGB1 release and then investigate the potential molecular mechanisms. In an experimental rat model of sepsis caused by CLP, salidroside administration significantly attenuated lung injury and reduced the serum HMGB1 level. In RAW264.7 cells, we investigated the effects of salidroside on LPS-induced HMGB1 release and then explored the underlying molecular mechanisms. We found that salidroside significantly inhibited LPS-induced HMGB1 release, and the inhibitory effect was correlated with the HMGB1 acetylation levels. Mechanismly, salidroside inhibits HMGB1 acetylation through the AMPK-SirT1 pathway. In addition, SirT1 overexpression attenuated LPS-induced HMGB1 acetylation and nucleocytoplasmic translocation. Furthermore, in SirT1 shRNA plasmid-transfected cells, salidroside treatment enhanced SirT1 expression and reduced LPS-activated HMGB1 acetylation and nucleocytoplasmic translocation. Collectively, these results demonstrated that salidroside might reduce HMGB1 release through the AMPK-SirT1 signalling pathway and suppress HMGB1 acetylation and nucleocytoplasmic translocation.