Accumulating evidence has indicated that Raf kinase inhibitor protein (RKIP) is involved in several intracellular signaling pathways; its abnormal expression is associated with tumor progression and metastasis in several human neoplasms. However, the role of RKIP in acute liver injury has remained elusive. In the present study, acute liver failure was induced by thioacetamide in mice, and locostatin was used to interfere with RKIP expression. It was found that RKIP expression was significantly inhibited by locostatin. Down-regulation of RKIP expression resulted in severe liver injury and extensive release of alanine aminotransferase and aspartate aminotransferase. In addition, reduced RKIP expression significantly enhanced the levels of reactive oxygen species and the content of pro-inflammatory factors such as tumor necrosis factor-α as well as interleukin-6 and -1β, and decreased the levels of nuclear factor E2-related factor-2 and heme oxygenase-1. Furthermore, down-regulation of RKIP promoted the activation of the nuclear factor-κB and extracellular signal-regulated kinase signaling pathways. In conclusion, the present study indicates an inverse correlation between RKIP level and the degree of hepatic injury, that is, a decrease in RKIP expression may exacerbate acute liver failure.