Background. HMGB1 acts as an important inflammatory mediator and is a potential therapeutic target for sepsis. Glycyrrhizin (GL), a natural triterpene glycoside derived from licorice, has been demonstrated to inhibit HMGB1 activity. The aim of this study is to explore how GL affects the HMGB1 signaling in sepsis. Methods. We used a CLP model of sepsis and in vitro LPS or HMGB1-treated NR8383 cells to examine the effects of GL on expression of HMGB1 and proinflammatory cytokines. Furthermore, we explored the effect of GL on interactions between HMGB1 and RAGE or TLR4 and the activations of NF-κB and MAPKs. Results. GL significantly decreased mortality and reduced serum levels of HMGB1 in vivo. GL also attenuated the release and expression of HMGB1 and proinflammatory cytokines. Direct stimulation by HMGB1 elevated the release of proinflammatory cytokines faster than LPS did and it was also inhibited by GL. Furthermore, GL blocked the interaction of HMGB1 with RAGE and TLR4 and suppressed the downstream MAPKs/NF-κB signaling pathway. Conclusion. GL may protect rats against sepsis by blocking the interaction of HMGB1 with cell surface receptors and HMGB1-mediated inflammatory responses.