Objective This paper was designed to assess the anti-tumor immunological effect and reveal the molecular mechanism of Astragaloside II in enhancing the anti-tumor immunological response via regulating CD45 PTPase. This study will provide a basis for the theoretical foundation of antitumor immunotherapy of Astragalus. Methods The H22 tumor-bearing mice was established to investigate the anti-tumor immunological effect of Astragaloside II. Mice were divided into three groups, including model group, Astragaloside II group, and Astragaloside II+Anti-CD45 Ab group. After 21 days of intragastric administration, the tumor tissue was isolated to assess tumor weight, tumor diameter and tumor volume. The lymphocyte cells proliferation from H22 tumor-bearing mice was detected by MTT method. The mRNA expression of Th1 cytokine, Th2 cytokine, and transcript factor T-bet were examined by q-PCR analysis. The expression of Th1 (CD4+ IFN- γ+ ), Treg (CD4+ Foxp3+ ) and Th17 (CD4+ IL-17+ ) were detected by flow cytometry. Results The tumor weight, tumor diameter and tumor volume in Astragaloside II group are more small than model group(P<0.05). Astragaloside II treatment significantly enhance the lymphocyte proliferation activity from H22 tumor-bearing mice in response to ConA, the transcriptional expression in Th1 and Th2, including IFN-γ, IL-2, IL-4, and T-bet were up-regulated. Meanwhile, IFN-γ expression was up-regulated in CD4+ T cells but down-regulated in T regulatory cells By Astragaloside II. Furthermore, anti-mouse CD45 Ab treatment intensely blocked the anti-tumor effect and lymphocyte proliferation activity which induced by Astragaloside. Compared with Astragaloside II group, IL-2, IFN-γ and IL-4 mRNA expression was decreased in Astragaloside II+Anti-CD45 Ab group. Anti-mouse CD45 Ab treatment intensely down-regulated IFN-γ expression in CD4+ T cells without significantly influencing Treg cells. Conclusion Astragaloside II might enhanced anti-tumor immunological response via regulating CD45 protein tyrosine phosphatase activity. This novel mechanism will provide a basis for the clinical application of Astragalus.