Background: Renal ischemia reperfusion injury (IRI) leads to acute kidney injury (AKI) and the death of tubular epithelial cells (TEC). The release of high-mobility group box-1 (HMGB1) and other damage-associated molecular pattern moieties from dying cells may promote organ dysfunction and inflammation by effects on TEC. Glycyrrhizic acid (GZA) is a functional inhibitor of HMGB1, but its ability to attenuate the HMGB1-mediated injury of TEC has not been tested. Methods/Results: In vitro, hypoxia and cytokine treatment killed TEC and resulted in the progressive release of HMGB1 into the supernatant. GZA reduced the hypoxia-induced TEC death as measured by annexin-V and propidium iodide. Hypoxia increased the expression of MCP-1 and CXCL1 in TEC, which was reduced by GZA in a dose-dependent manner. Similarly, the HMGB1 activation of effector NK cells was inhibited by GZA. To test the effect of HMGB1 neutralization by GZA in vivo, mice were subjected to renal IRI. HMGB1 protein expression increased progressively in kidneys from 4 to 24 h after ischemia and was detected in tubular cells by 4 h using immunohistochemistry. GZA preserved renal function after IRI and reduced tubular necrosis and neutrophil infiltration by histological analyses and ethidium homodimer staining. Conclusions: Importantly, these data demonstrate for the first time that AKI following hypoxia and renal IRI may be promoted by HMGB1 release, which can reduce the survival of TEC and augment inflammation. Inhibition of the interaction of HMGB1 with TEC through GZA may represent a therapeutic strategy for the attenuation of renal injury following IRI and transplantation.