Objective To explore the immunomodulatory effects of natural killer (NK) cells on the CD4⁺T cell subset in mice with cerebral malaria. Methods Female C57BL/6 mice were randomly divided into the health control group, infection group and NK elimination group, 14 mice in each. The mice in the infection group and NK elimination group were injected intraperitoneally with 1 × 10⁶ Plasmodium berghei ANKA (PbA)-infected red cells. The mice in NK elimination group were intraperitoneally injected with anti-Asialo GM-1(15 μl), a blocking antibody against NK cells, on day 1 before and day 2 after infection, while the mice in the health control and infection groups were injected with equal volumes of PBS. From day 3 on post-infection, blood was collected from the tail vein to make blood smears for examining parasitemia, and death of mine was recorded. On days 3 and 5 after infection, 3 mice were sacrificed in each group. The spleens were taken to make single cell suspensions for assaying Th1 type cells and regulatory T cells (Tregs) by flow cytometry. The spleen cells were cultured in vitro for 48 h, then the culture supernatant was collected to assay the levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin 10 (IL-10) by ELISA. On day 6 after infection, 3 mice each group received Evans blue (EB) intravenously, brain tissues were incubated in vitro for 48 h, supernatant was collected and the EB content passing through the blood-brain barrier of the mice was detected by microplate reader. Results In the infection group, mice deaths began to occur on day 6 after infection, accompanied by neurological symptoms, and all died on day 8. In the NK elimination group, deaths began to occur on day 7 after infection, and all mice died on day 12. On day 7 after infection, mice in the NK elimination group had significantly higher parasitemia (5.2%-10.5%) than those in the infection group (2.4%-5.0%) (P < 0.05). On day 5 after infection, the percentage and absolute counts of Th1 type cells were (2.65 ± 0.06)% and (2.09-2.25) × 10⁶in the infection group, which were significantly higher than those in the health control group (1.37 ± 0.02)% and (0.41-0.47) × 10⁶ (P < 0.01). and those in the NK elimination group [(1.82 ± 0.07)% and (1.48-1.86) × 10⁶] were significantly lower than those in the infection group (P < 0.01 or P < 0.05). The percentages of Tregs on days 3 and 5 in the infection group were(1.21 ± 0.06)% and (1.70 ± 0.13)%, respectively, which were significantly higher than those in the health control group[(0.90 ± 0.01)% and (0.91 ± 0.02)%] (P < 0.01). The absolute counts of Tregs in the infection group on day 3 and 5 post-infection were (0.63-0.73) × 10⁶ and (1.39-1.62) × 10⁶, respectively, which were higher than those in the health control group[(0.27-0.31) × 10⁶ and (0.47-0.56) × 10⁶] (P < 0.01); the percentages of Tregs in the NK elimination group were (1.86 ± 0.07)% and (2.15 ± 0.04)%, and the absolute counts were (0.77-0.90) × 10⁶ and (1.68-2.15) × 10⁶, respectively, all significantly higher than those in the infection group (P < 0.01 or P < 0.05). On days 3 and 5 after infection, the concentrations of IFN-γ in the cultural supernatant of splenocytes in the infection group were (790.75 ± 84.80) and (989.58 ± 199.59) pg/ml, respectively, and the TNF-α concentrations were (2 637.47 ± 283.50) and (3 124.58 ± 964.70) pg/ml, respectively, which were higher than those in the health control group [(73.69 ± 16.67) and (75.19 ± 15.97) pg/ml, (290.01 ± 187.46) and (290.51 ± 186.76) pg/ml respectively] (P < 0.01 or P < 0.05). In the NK elimination group, the IFN-γ concentrations were (15.83 ± 4.27) and (266.63 ± 108.62) pg/ml, and the TNF-α concentrations were (165.89 ± 71.02) and (842.77 ± 311.94) pg/ml, respectively, which were lower than those in the infection group (P < 0.01 or P < 0.05). On day 3 after infection, the IL-10 concentration in the NK elimination group was (3 588.20 ± 1 436.38) pg/ml, which was higher than that in the infection group [(1 255.77 ± 190.01) pg/ml] (P < 0.05). On day 5 after infection, the IL-10 concentration in the infection group was (4 991.36 ± 1 030.89) pg/ml, which was higher than that in the health control group as (848.50 ± 501.79) pg/ml (P < 0.05). In the NK elimination group, the IL-10 concentration was (9 317.95 ± 1 077.89) pg/ml, which was higher than that in the infection group (P < 0.05). On day 6 after infection, the EB content in the brain tissue of the infection group was (4.02 ± 0.10) μg/ml, which was higher than that in the health control group [(2.09 ± 0.06) μg/ml] (P < 0.05), and the EB content in the NK elimination group was (3.14 ± 0.02) μg/ml, which was lower than that in the infection group (P < 0.05). Conclusion The elimination of NK cells weakens the Th1 immune response, enhances the Tregs immune response, reduces the blood-brain barrier permeability and prolongs the survival time of mice with cerebral malaria.