Objective To explore the effects of pyrimethamine, an anti-Toxoplasma gondii drug, on folate level and immune function of macrophages. Methods RAW264.7 cells were seeded in 96-well plates (1 × 10⁴ cells/well) and treated with 0 (control), 0.01, 0.05, 0.10, 0.50, 1.00, 5.00, 10.00, 50.00, 100.00, 500.00 and 1 000.00 μmol/L pyrimethamine for 24 h, respectively, followed by CCK8 assay. RAW264.7 cells were seeded in 6-well plates (5 × 10⁵ cells/well), and infected with T. gondii tachyzoites for 3 h, followed by treatement with 0.10, 1.00, 10.00 and 100.00 μmol/L pyrimethamine for 24 h, respectively. The expression of dihydrofolate reductase (DHFR) in T. gondii tachyzoites was detected by qRT-PCR. Total RNA of RAW264.7 cells was extracted by TRIzol reagent. The expression of DHFR and inflammatory genes in macrophages was detected by qRT-PCR, the protein abundance of DHFR was detected by Western blotting and intracellular folate levels were detected by Automated Chemiluminescence Systems. The amino acid sequences of DHFR from T. gondii tachyzoites and from RAW264.7 cells were compared by BLAST. RAW264.7 cells were cultured in folate-free medium for 48 h, and then treated with 100 μmol/L pyrimethamine for 24 h to detect the expression of inflammatory genes by qRT-PCR. The comparison between two groups was performed by t-test, and multiple comparison was performed by one-way ANOVA. Results CCK8 detection showed that the inhibition rate of pyrimethamine on cell activity was in a dose-dependent manner, which could induce slight proliferation of macrophages at 0.01-0.10 μmol/L, while inhibited cell proliferation at the concentration higher than 0.10 μmol/L. qRT-PCR showed that the mRNA expression of DHFR in T. gondii tachyzoites in the 0.10, 1.00, 10.00 and 100.00 μmol/L groups were 1.190 ± 0.054, 1.460 ± 0.206, 0.468 ± 0.077 and 0.399 ± 0.073 respectively, showing mRNA expression in the 1.00 μmol/L group being higher than that in the control group, and the level in the 10.00 and 100.00 μmol/L groups lower than that in the control group (P < 0.01). Meanwhile, the mRNA expression of DHFR in the RAW264.7 cells were 0.789 ± 0.505, 1.820 ± 0.119, 2.120 ± 0.140 and 2.080 ± 0.189 respectively, higher in the 1.00, 10.00 and 100.00 μmol/L groups than those in the control group (P < 0.05 or 0.01). Western blotting results showed that the protein abundance of DHFR in macrophages in the 100.00 μmol/L group was significantly higher than that in the control group. BLAST results showed that the homology between tachyzoite DHFR and mouse macrophage DHFR was only 37.0%. Using Automated Chemiluminescence Systems, we found that the intracellular folate level of macrophages in the 100.00 μmol/L group was (10.600 ± 2.930) nmol/L, which was much higher than that in the control group (P < 0.01). After treatment with 100 μmol/L pyrimethamine, the mRNA expression of TNF-α, iNOS, Arg-1 and IL-1β were 2.230 ± 0.100, 11.800 ± 1.350, 3.610 ± 0.256 and 7.810 ± 0.987 respectively, which were all higher than those in the control group (P < 0.05 or 0.01). After treatment with 100 μmol/L pyrimethamine, the mRNA levels of TNF-α, iNOS, Arg-1 and IL-1β in the folate-free culture group were 2.460 ± 0.026, 1.330 ± 0.091, 0.974 ± 0.141 and 0.997 ± 0.018 respectively. The mRNA levels of iNOS, Arg-1 and IL-1β in the folate-free group were much lower than those in the normal folate group (P < 0.05 or 0.01), while the opposite was found for TNF-α (P < 0.01). Conclusion Pyrimethamine could increase the intracellular folate level of macrophages by promoting the expression of DHFR in mice, which further induces the inflammatory gene expression of macrophages.