Abstract: OBJECTIVE To investigate the mechanism of killing effect of cordycepin on tumor-associated CD4⁺T lymphocyte tumor in vitro. METHODS Tumor-associated CD4⁺T-lymphocytes were isolated from xenograft tumors. MTS assay was used to study the effect of cordycepin on the proliferation of tumor-associated CD4⁺T-lymphocytes; LDH method was used to analyze the killing effect of tumor-associated CD4⁺T lymphocytes treated with cordycepin. For the co-incubation of tumor cells， the cytotoxicity of the supernatants of the cells incubated with CD4⁺T lymphocytes and tumor cells was detected by ELISA， and Western blot was used to study the related protein in the PI3K/AKT pathway. RESULTS Cordycepin could promote the proliferation and killing effect of tumor-associated CD4⁺T lymphocytes in a dose-dependent manner， and cordycepin reduced PD-1 protein and enhanced PI3K/AKT pathway-associated protein expression and promote IL-2 and IFN-γ expression in tumor-associated CD4⁺T lymphocytes. CONCLUSION Cordycepin can promote the proliferation and killing effect of tumor-associated CD4⁺T lymphocytes and its mechanism may be related to the inhibition of tumor-immune checkpoint PD-1.