Nuclear factor κB (NF-κB) has a central role in coordinating the expression of a wide variety of genes that control immune responses and is also recognized as an antiapoptotic transcription factor. Here, we focused on the role of the NF-κB signaling pathway in the interaction between inflammatory cells and hepatocytes in liver inflammation. We found that pretreatment of mice with adenoviruses expressing a mutant form of the inhibitor κB superrepressor (Ad5IκB), a NF-κB inhibitor, reduced the migration of inflammatory cells and cytokine and chemokine expression in the liver 12 hours after a single intravenous injection of an anti-CD40 antibody (αCD40) compared with mice infected with control adenoviruses (Ad5LacZ). We also confirmed reductions in cytokine production by macrophages, T cells, and natural killer (NK) cells in the liver of Ad5IκB-treated mice by FACS analysis. However, αCD40 treatment in Ad5IκB-infected mice induced elevation of serum alanine aminotransferase at 24 hours, and the liver injury was associated with massive hepatocyte apoptosis. Furthermore, interferon gamma (IFN-γ) production by NK cells and T cells was increased and stimulated tumor necrosis factor alpha (TNF-α) production by macrophages in the Ad5IκB-infected liver. Moreover, the liver injury was completely suppressed by the administration of anti–IFN-γ and anti–TNF-α. These results suggest that inhibition of NF-κB activity suppressed αCD40-induced liver inflammation at an early phase, resulting in a reduction in cytokine and chemokine production, whereas it sensitized hepatocytes to TNF-α–induced apoptosis and exacerbated liver injury at the late phase. In conclusion, NF-κB exerts pivotal activities at inflammatory sites, and caution should be exercised in NF-κB–targeted therapy of liver disease. (HEPATOLOGY 2004;40:1180–1189.)