Objective To observe the effects of Grifola frondosa polysaccharide on T cell subsets and Th1/Th2 subsets of spleen deficiency mice, and investigate its immunoregulation mechanism. Methods Forty-eight KM mice were divided into 6 groups:normal group, spleen deficiency group, positive group (lentinan), three dosage groups of Grifola frondosa polysaccharide, 8 mice in each group. The mice were injected corresponding drug intraperitoneally for 10 days. Then mice in each group were detected CD3+, CD4+ and CD8+T cell by flow cytometry. IFN-γ and IL-4 levels were detected by ELISA. Results The high dose of Grifola frondosa polysaccharide could significantly increase the CD4+T lymphocytes percentage in peripheral blood (P<0.05). Medium and high dose of Grifola frondosa polysaccharide could significantly increase CD4+/CD8+T cell ratio (P<0.01) and CD3+T lymphocyte percentage (P <0.01) of spleen deficiency mice. Medium and high dose of grifola frondosa polysaccharide could significantly increase IFN-γ level in serum of spleen deficiency mice (P<0.01), and high dose of Grifola frondosa polysaccharide could decrease IL-4 level in serum of spleen deficiency mice (P<0.01). Conclusion Grifola frondosa polysaccharide can improve the decreased CD4+/CD8+T cell ratio which caused by spleen deficiency, and promote the transformation of Th1 to Th2, thus treating spleen deficiency syndrome.