Liver lymphocytes are enriched in natural killer (NK) cells, and activation of NK cells by injection of polyinosinic-polycytidylic acid (poly I:C) inhibits liver regeneration in the partial hepatectomy model via production of IFN-γ. However, the role of NK cells in liver regeneration in a model of carbon tetrachloride (CCl₄)-induced liver injury remains unknown. In this study, we investigated the effect of activation of NK cells induced by poly I:C on liver regeneration in the CCl₄ model. Administration of poly I:C suppressed liver regeneration in CCl₄-treated mice. Depletion of NK cells but not Kupffer cells or T cells restored liver regeneration in poly I:C/CCl₄-treated mice. Poly I:C and CCl₄ cotreatment synergistically induced accumulation of NK cells in the liver and NK cell production of IFN-γ and tumor necrosis factor (TNF)-α. Serum levels of these two cytokines were also synergistically induced after poly I:C and CCl₄ treatment. Finally, blockage of TNF-α but not IFN-γ restored liver regeneration in poly I:C/CCl₄-treated mice. Taken together, these findings suggest that poly I:C treatment inhibits liver regeneration in the CCl₄-induced liver injury model via induction of NK cell production of TNF-α. Copyright © 2010 the American Physiological Society.