Extensive in vivo and vitro studies showed that the Toll-like receptor (TLR) 7/8 agonist resiquimod(R848) had potent Th1 adjuvant activity. The role of R848 in Th1 induction is dependent on R848 induction of dendritic cell (DC)maturation and the promotion of IL-12 production. Here We demonstrated that R848 produced stronger Th1 induction than two common TLR agonists and revealed that the stronger Th1 response induced by R848 was attributed to DC-NK interaction. R848 induced IFN-γ+CD8+ T cell and CD8+T cell accumulation in lymph nodes to a greater degree in vivo than LPS(Lipopolysaccharide) and CPG-C(Class C CPG ODN).Increased natural killer(NK) cell migration to lymph nodes occurred in R848-immunized mice. R848 more potently activated DC and NK cells in vitro. R848-activated DCs enhancedCD8+ T cell proliferation and increased IFN-γ+CD8+ T cells with the assistance of NK cells. Further study demonstrated that R848-activated DCs increased chemokine-CXCL9 production and induced more NK cell migration than DCs activated with the other two TLR agonists. Taken together, our results show that R848 is a new potent Th1 immune adjuvant that may not merely depend on DC activation, and NK cells must also be considered. With the growing immunological understanding of the effects of R848, these findings provide a new perspective for R848 application in clinical investigations.