Concanavalin A (Con A), a widely used murine T cell stimulator, could induce T cell-mediated hepatitis when used in an abundant dosage. However, the immune-modulating role of non-hepatotoxic dosage of Con A in hepatic immune responses remains obscure. In order to evaluate the effect of non-hepatotoxic Con A on hepatic NK cells, pretreatment of mice with low dose of Con A was performed on polyinosinic–polycytidylic acid (poly I:C) [toll-like receptor-3 (TLR-3) ligand]-induced NK cell-mediated hepatitis. The role of the Con A-pre-activated T cells in NK cell function was then determined. The results demonstrated that Con A pretreatment aggravated poly I:C-induced liver injury with elevation of serum transaminases, hepatic necrosis and serum IFN-γ level. In parallel to the enhanced accumulation of activated NK cells into liver, the natural cytotoxicity and IFN-γ production by hepatic NK cells were significantly augmented. Moreover, depletion of T cells suppressed the aggravating effect of Con A pretreatment. Collectively, the aggravation of poly I:C-induced hepatitis by Con A pretreatment may be due to the generation of more vigorous NK cells which is dependent on the presence of T cells. This investigation will help to explain the synergistic effects of NK and T cells in liver inflammatory injury.