Background and AimAcute liver failure (ALF) is a fatal clinical syndrome of severe hepatic dysfunction. Chemokines promote liver diseases by recruiting and activating immune cells. We aimed to investigate the role of C–C chemokine ligand 25 (CCL25) in ALF.MethodsAn ALF mouse model induced by D‐galactosamine/lipopolysaccharide was evaluated through liver hematoxylin and eosin staining and serum transaminase and cytokine measurement. CCL25 expression in serum was analyzed by ELISA and in liver by immunohistochemical staining and western blot. C–C chemokine receptor 9 (CCR9)‐expressing cells in the liver were identified by immunofluorescence staining. The effects of anti‐CCL25 on ALF were evaluated in vivo. Cytokine expression and migration of CCL25‐stimulated RAW264.7 macrophages were studied. We also investigated the role of anti‐CCL25 and BMS‐345541, an NF‐κB signaling inhibitor, in vitro. NF‐κB activation was assessed via western blot, and p65 nuclear translocation was detected using cellular immunofluorescence.ResultsALF mice showed severe histological damage and high serum levels of aminotransferase and inflammatory cytokines. Elevated CCL25 and NF‐κB activation was observed in vivo. CCR9 was expressed on macrophages in ALF mouse liver. ALF was suppressed after anti‐CCL25 treatment, with significant NF‐κB inhibition. In vitro, CCL25 induced strong migration and cytokine release in RAW264.7 macrophages, which were eliminated by anti‐CCL25 and BMS‐345541. Furthermore, the NF‐κB activation and p65 nuclear translocation induced by CCL25 were also inhibited by anti‐CCL25 and BMS‐345541.ConclusionCCL25 contributes to ALF development by inducing macrophage‐mediated inflammation via activation of the NF‐κB signaling.