Aberrant expression of the chemokine CXC chemokine ligand (CXCL)10 has been linked to the severity of hepatitis C virus (HCV)-induced liver injury, but the underlying molecular mechanisms remain unclear. In this study, we describe a yet-unknown proapoptotic effect of CXCL10 in hepatocytes, which is not mediated through its cognate chemokine receptor, but the lipopolysaccharide receptor Toll-like receptor 4 (TLR4). To this end, we investigated the link of CXCL10 expression with apoptosis in HCV-infected patients and in murine liver injury models. Mice were treated with CXCL10 or neutralizing antibody to systematically analyze effects on hepatocellular apoptosis in vivo. Direct proapoptotic functions of CXCL10 on different liver cell types were evaluated in detail in vitro. The results showed that CXCL10 expression was positively correlated with liver cell apoptosis in humans and mice. Neutralization of CXCL10 ameliorated concanavalin A–induced tissue injury in vivo, which was strongly associated with reduced liver cell apoptosis. In vitro, CXCL10 mediated the apoptosis of hepatocytes involving TLR4, but not CXC chemokine receptor 3 signaling. Specifically, CXCL10 induced long-term protein kinase B and Jun N-terminal kinase activation, leading to hepatocyte apoptosis by caspase-8, caspase-3, and p21-activated kinase 2 cleavage. Accordingly, systemic application of CXCL10 led to TLR4-induced liver cell apoptosis in vivo. Conclusion: The results identify CXCL10 and its noncognate receptor, TLR4, as a proapoptotic signaling cascade during liver injury. Antagonism of the CXCL10/TLR4 pathway might be a therapeutic option in liver diseases associated with increased apoptosis. (HEPATOLOGY 2013)