Abstract In breast cancer therapy, as the leading cause of death in women, besides chemo-radiotherapy, immunotherapy has been increasingly used. PD-1/PD-L1 axis blockade primarily acts on T lymphocytes, the main effectors of acquired immune response. NK cells, which are part of the innate immune response, also play a role in the anti-tumor response through the blockade of this signaling pathway. The study was conducted to examine the effects of anti-PD-1 therapy on NK and T cells in mouse breast cancer. Female BALB/c mice were used, divided into two groups, one with induced breast cancer and one treated with anti-PD-1 antibody. Breast cancer cell line was used to induce the cancer, and the anti-PD-1 antibody was applied intraperitoneally. Cell populations in spleen and tumor microenvironment were examined using flow cytometry. Data were statistically analyzed using SPSS. The percentage of NK cells expressing FasL, NKG2D, and IFN-γ is significantly higher in spleen and tumor-infiltrating NK cells upon anti-PD-1 therapy, while the expression of inhibitory markers Foxp3 and IL-10 in regulatory NK cells is significantly lower. The percentage of T lymphocytes expressing CD107a and IL-17 is significantly higher in the spleen, while a higher number of T lymphocytes expressing CD69 is present in the tumor microenvironment. The study suggests that anti-PD-1 therapy can activate NK and T cells, and improve anti-tumor immune response in breast cancer. Further research is needed to understand the interplay between these cells during PD-1 blockage.