The role of hepatic CD69+ natural killer (NK) cells in virus-induced severe liver injury and subsequent hepatic failure is not well defined. In this study, a mouse model of fulminant liver failure (FHF) induced by murine hepatitis virus strain 3 (MHV-3) was used to study the role of hepatic CD69+ NK cells in the development of FHF. The CD69 expression in NK cells in the liver, spleen, bone marrow and peripheral blood was detected by using flow cytometry. The correlation between the CD69 level in hepatic NK cells and liver injury was studied. The functional marker (CD107a), and activating and inhibitory receptor (NKG2D and NKG2A) expressed on CD69+ NK cells and CD69-NK cells were detected by using flow cytometry. Pro-inflammatory cytokines (IL-9, IFN-gamma and TNF-alpha) were also examined by using intracellular staining. After MHV-3 infection, the number of CD69+ NK cells in the liver of BALB/cJ mice was increased markedly and peaked at 72 h post-infection. Similar changes were also observed in the spleen, bone marrow and peripheral blood. Meanwhile, the CD69 expression in hepatic NK cells was highly correlated with the serum level of ALT and AST. The expression of CD107a and NKG2D, as well as the production of TNF-alpha, IFN-gamma and IL-9 in hepatic CD69+ NK cells was all significantly up-regulated during 48-72 h post-infection. In contrast, the NKG2A expression was increased in hepatic CD69-NK cells but not in CD69+ NK cells. These results suggested that hepatic CD69+ NK cells play a pivotal role in the pathogenesis of FHF by enhancing degranulation and cytotoxic ability of NK cells and increasing the production of pro-inflammatory cytokines.