Studies have shown that the HMGB1-TLR4 (High-mobility group protein B1, toll-like receptor 4) pathway participates in renal ischemic reperfusion injury (IRI) and that dexamethasone (DEX) could protect the kidney against IRI. This study aims to examine the protective effects of DEX on renal IRI and further explore the possible mechanism of action. During mouse renal IRI, HMGB1-TLR4 signals changed markedly including HMGB1 translocation and TLR4 up-regulation, resulting in histological damage and an increase in MPO expression. Treatment with DEX markedly decreased the damage to renal function (serum Cr and BUN; kidney KIM-1 expression) and the histological pathology of the kidney after renal IRI. The activation of GR by DEX did not suppress p38 and JNK activity but inhibited ERK phosphorylation. Treatment with DEX also attenuated I kappa B-alpha phosphorylation and further reduced NF-kappa B expression in the nucleus by decreasing acetylation of the p65 subunit. Furthermore, the HMGB1-TLR4 inflammatory pathway was inhibited via the attenuated translocation of HMGB1 from the nucleus to the cytoplasm and the down-regulation of TLR4 expression through DEX treatment. The inhibition of HMGB1 translocation may interact with acetyltransferase and attenuate HMGB1 acetylation. As a result, the levels of cytokines (TNF-alpha, IL-6, and IL-1 beta) were down-regulated and inflammatory cell infiltration after renal IRI was attenuated by treatment with DEX. This study demonstrated that the HMGB1-TLR4 pathway may play a critical role in renal IRI. DEX may attenuate renal IRI by suppressing ERK and NF-kappa B activation, followed by attenuating the HMGB1-TLR4 pathway through inhibiting acetyltransferases.