Increasing evidence suggests the contribution of natural killer (NK) cells to pathogenesis of human hepatitis, but the detailed mechanisms have yet to be clearly elucidated. In this study, injection of polyinosinic:polycytidylic acid (poly I:C) and D-galactosamine (D-GalN) was used to establish a novel murine fulminant hepatitis model: results showed that predepletion of either NK cells or Kupffer cells could completely abolish the liver injury. Injection of poly I:C/D-GalN into mice could promote tumor necrosis factor-α production and surface retinoic acid early inducible-1 (Rae1) protein expression by Kupffer cells, which then activated NK cells to produce interferon-γ via NKG2D-Rae1 recognition. NK cell–derived interferon-γ and Kupffer cell–derived tumor necrosis factor-α synergistically mediated the severe liver injury. Moreover, Kupffer cell–derived interleukin-12 and interleukin-18 were also found to improve cross talk between NK cells and Kupffer cells. Conclusion: These results provide the first in vivo evidence that NKG2D/ligand interaction is involved in the synergic effects of NK cells and Kupffer cells on acute liver injury. (HEPATOLOGY 2009.)