Objectives: To analyze the effects of TLR4 on the expression of the HMGB1, MHC-I and downstream cytokines IL-6 and TNF-alpha, and to investigate the biological role of the TLR4-HMGB1 signaling pathway in the development of the autoimmune myositis. Methods: We built mice models with experimental autoimmune myositis (EAM) and used the inverted screen experiment to measure their muscle endurance; we also examined inflammatory infiltration of muscle tissues after HE staining; and we assessed the expression of MHC-I using immunohistochemistry. In addition, peripheral blood mononuclear cells (PBMC) were extracted and flow cytometry was utilized to detect the effect of IFN-gamma on the expression of MHC-I. Furthermore, PBMCs were treated with IFN-gamma, anti-TLR4, anti-HMGB1 and anti-MHC-I. Real-time PCR and western blotting were employed to examine the expressions of TLR4, HMGB1 and MHC-I in different groups. The ELISA method was also utilized to detect the expression of the downstream cytokines TNF-alpha and IL-6. Results: The expressions of TLR4, HMGB1 and MHC-I in muscle tissues from mice with EAM were significantly higher than those in the control group (all P < 0.05). After IFN-gamma treatment, the expressions of TLR4, HMGB1, MHC-I, TNF-alpha and IL-6 in PBMCs significantly increased (all P < 0.05). The treatment of anti-TLR4, anti-HMGB1 and anti-MHC-I could significantly downregulate the expression of MHC-I (all P < 0.05). In addition, anti-TLR4 and anti-HMGB1 significantly reduced the expression of TNF-alpha and IL-6 (all P < 0.05). Conclusions: The TLR4-HMGB1 signaling pathway affects the process of autoimmune myositis inflammation by regulating the expression of MHC-I and other pro-inflammatory cytokines. (C) 2016 Elsevier B.V. All rights reserved.