Objective To evaluate the regulatory effects of Astragalus polysaccharide (APS) on macrophage polarization and NK cell-mediated anti-tumor responses in mice. Methods C57BL/ 6 mice were injected intraperitoneally with APS once a day for seven consecutive days. Activation of immune cells was then induced by intraperitoneal injection of polyinosinic-polycytidylic acid (Poly I : C) 24 h after the APS intervention. Peritoneal macrophages were collected 24 h after induction to analyze the status of polari-zation and the production of nitric oxide (NO). Cytotoxicity and exocytosis of activated NK cells were meas-ured to assess the effector functions of these cells. NK cell activities induced by NKG2D were studied in the absence of the whole JNK or JNK2 signaling pathway. Results Intraperitoneal injection of APS promoted the polarization of macrophages induced by tumor cells in mice, and enhanced the cytotoxicity of NK cells to tumor cells. However, APS was in need of the involvement of appropriate stimulatory factors to have regula-tory effects. Complete inhibition of JNK signaling pathway dramatically reduced the effector functions of NK cells, which could not be recovered by APS administration. Conclusions APS was involved in the regula-tion of anti-tumor innate immunity through enhancing the M1-polarization of macrophages and improving the effector functions of NK cells. This study might to some extent elucidate the mechanism of APS in immune regulation and anti-tumor immunity.