Objective This paper was designed to reveal the new mechanism on ASI Ⅱ triggered CD4+T cells activation via regulating CD45 molecular and provide a basis for the theoretical foundation of antitumor immunotherapy of Astragalus. Methods The CD4+T cells were randomly divided into negative group, stimulated control group, ASIⅡgroup, CD45 inhibitor group, and the combination of ASIⅡ and CD45 inhibitor group. Besides negative group, the cells from other groups were activated by anti-CD3/CD28 antibody. ASIⅡgroup was treated with 10 nmol/L ASIⅡ, CD45 inhibitor group was treated with 0.8 μmol/L CD45 inhibitor, and the combination group were treated with 10 nmol/L ASIⅡ and 0.8 μmol/L CD45 inhibitor. After 36h culture, the proliferation of CD4+T cells was detected by Ki-67 intracellular staining assay. Cytokine production of Th1 and Th2 were examined ELISA method. The proportion of surface marker (CD44 and CD25) and Th1 intracellular cytokines (IFN-γ) were detected by flow cytometry. Results Compared with stimulated group, Astragaloside Ⅱ group in CD4+Ki67+T positive proportion (5.37% ± 0.92% vs. 1.19% ± 0.23%), in CD4+CD25+ positive proportion (50.23% ± 4.65 % vs. 15.89% ± 1.13%), in CD4+CD44+ positive proportion (33.16% ± 6.08% vs. 15.36% ± 1.45%), in CD4+IFN-γ+ positive proportion (1.42% ± 0.44 % vs. 0.38% ± 0.06%) were significntly increased. And the secretion of IFN-γ, IL-4 and IL-2 in ASI Ⅱ group were higher than stimulated group. The anti-mouse CD45 Ab treatment markedly blocked the proliferation and Th1 cytokines production which induced by ASI Ⅱ. Furthermore, the anti-mouse CD45 Ab treatment significantly decreased the expression of surface marker (CD44 and CD25). Conclusions Activating CD45 protein tyrosine phosphatase may be involved in ASI Ⅱ triggered CD4+T cells activation. This study will provide a basis for antitumor immunotherapy of Astragalus. Key words: Astragaloside; CD45 molecule; CD45 inhibitor; Cell proliferation; Interferon-gamma; Mice