Objective To investigate the effect of poly(I:C)-adjuvanted E7(44-62) on natural killer cells-mediated anti-tumor immunity. Methods NK92 cells were treated with medium (C group), poly(I:C) [poly(I:C) group] or poly(I:C)-adjuvanted E7(44-62) (combined group). Then, toll-like receptor 3 expression, TNF-α and INF-γ mRNA levels and tumor cell-killing activity were measured before and after treatment. Results One hour after treatment, the toll-like receptor 3 expression in the combined group was obviously higher than those of C group and poly(I:C) group. One hour after treatment, the level of TNF-α mRNA in the combined group was significantly higher than those of C group and poly(I:C) group (all P<0.01); the level of INF-γ mRNA in the combined group was significantly higher than those of C group and poly(I:C) group (all P<0.05). One day after treatment, the tumor cell-killing activity in the combined group was significantly higher than those of C group and poly(I:C) group (all P<0.01). Conclusions Poly(I:C)-adjuvanted E7(44-62) can enhance ntural killer cells-mediated anti-tumor immunity by increasing the expression of Toll-like receptor 3, TNF-α and INF-γ gene.