Acute-on-chronic liver failure (ACLF) is a disease in which patients convert from chronic liver disease to acute liver failure triggered by multiple factors, with the characteristics of rapid development and high lethality. Centering in ACLF, the study herein explored the effect of high mobility group box 1 protein (HMGB1), toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE) in the progression of ACLF. ACLF patients admitted to our hospital from April 2017 to April 2020 were selected as the research objects. Their serum indicators were measured with the Siemens biochemical line instrument; relevant indicators of coagulation system were measured with the ACL TOP/TOP700 instrument; the expression levels of HMGB1, TLR4 and RAGE in patients' serum were measured by Enzyme linked immunosorbent assay (ELISA); the in vivo study was conducted with 42 male Wistar rats; 6 normal rats were in the control group, and the model of liver cirrhosis was established with 28 rats by subcutaneous injection of human serum albumin (HSA), then the 28 rats with liver fibrosis above grade III were randomly divided into the ACLF group (n = 11), HMGB1 inhibitor (glycyrrhizic acid) group (n = 11) and liver cirrhosis group (n=6); among them, the rats in the ACLF group accepted intraperitoneal injection of 400 mg/kg of D-GalN and 100 ug/kg of LPS; and before the same intraperitoneal injection as the ACLF group, the rats in the HMGB1 inhibitor group were administered intraperitoneally with 50 mg/kg of glycyrrhizic acid daily for 3 days; and the equal doses of normal saline were injected into the abdominal cavity in rats in the control group and liver cirrhosis group respectively; and the levels of HMGB1, TLR4 and RAGE in rats' liver tissue were analyzed by immunohistochemistry and Western blot. Compared with the ineffective group, the international normalised ratio (INR) of serum, urea and plasma prothrombin time (PT) in patients of the effective group were significantly reduced; the levels of serum HMGB1, RAGE and TLR4 in patients of the effective group were significant different after treated for 10 days, and were significantly lower than those of the ineffective group after the end of treatment; compared with the normal control group and the liver cirrhosis group, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and PT were significantly increased in the ACLF group (p < 0.01), and obvious improvement was showed in the ACLF rats treated with glycyrrhizic acid (p < 0.01); compared with the normal control group and the liver cirrhosis group, ACLF rats had serious hepatic cell injury (p < 0.01); hematoxylin-eosin (HE) staining results showed that the rats in the ACLF group had serious hepatic cell injury, and those treated with glycyrrhizic acid obviously improved (p < 0.01); in addition, the immunohistochemistry results showed that ACLF rats had significantly increased levels of HMGB1, TLR4 and RAGE in the liver, and those treated with glycyrrhizic acid obviously improved (p < 0.01). HMGB1, TLR4 and RAGE were closely related to the progression of ACLF, which can be used