High-mobility group box 1 (HMGB1), an abundant nuclear protein that triggers host immune responses, is an endogenous danger signal involved in the pathogenesis of various infectious agents. However, its role in hepatitis C virus (HCV) infection is not known. Here, we show that HMGB1 protein is translocated from the nucleus to cytoplasm and subsequently is released into the extracellular milieu by HCV infection. Secreted HMGB1 triggers antiviral responses and blocks HCV infection, a mechanism that may limit HCV propagation in HCV patients. Secreted HMGB1 also may have a role in liver cirrhosis, which is a common comorbidity in HCV patients. Further investigations into the roles of HMGB1 in the diseases caused by HCV infection will shed light on and potentially help prevent these serious and prevalent HCV-related diseases.