Objective To investigate the effect of Toxoplasma gondii infection during pregnancy on the expression of T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) in decidual immune cells and differential relationships. Methods Pregnant mice were assigned into the wild-type (WT) pregnant control group, the WT pregnant infection group, and the Tim-3^-/- pregnant infection group, of 20 mice each group. Mice in two infection groups were intraperitoneally inoculated with 250 tachyzoites of the T. gondii RH strain suspended in 250 μl of diluents at gestational ages of 8 days, while mice in the control group received an equal volume of physiological saline. The mental state of pregnant mice was observed in each group at gestational ages of 14 days, and all pregnant mice were sacrificed with euthanasia, dissected, recorded for pregnancy outcomes and calculate for the proportion of abnormal fetuses. Placentas and fetuses were harvested from pregnant mice and weighed, and the differences in adverse pregnancy outcomes were compared among three groups. Decidual tissues were collected from healthy pregnant women undergoing voluntary termination of pregnancy in the first trimester, and decidual mononuclear cells were isolated using density gradient centrifugation and divided into a control group and an infection group, with 1 × 10⁷ cells per group. Cells in the infection group were challenged with T. gondii at a cell-to-parasite ratio of 3 ∶ 1, whereas cells in the control group were treated with an equal volume of T. gondii-free culture media. Cells in two groups were harvested 20 hours post-incubation, and the expression of Tim-3 was detected on decidual natural killer (dNK) cells, decidual macrophages (dMϕ), decidual myeloid-derived suppressor cells (dMDSCs), and decidual dendritic cells (dDCs) using flow cytometry. dNK cells, dMϕ, dMDSCs and dDCs were further sorted from decidual mononuclear cells with immunomagnetic beads, and each cell type was subdivided into an infection subgroup and a control subgroup. Cells in the infected subgroups were infected with T. gondii at a cell-to-parasite ratio of 3∶1, and those in control subgroups were incubated with an equal volume of parasite-free culture media. Cells were harvested 20 hours post-incubation for protein purification, and Tim-3 expression in each type of decidual immune cells was determined using Western blotting assay. Between-group differences were tested for statistical significance with independent-sample t-test for in vivo experiments or paired-sample t-test for in vitro experiments. Results The placental [(83.13 ± 9.13) mg vs. (105.18 ± 9.64) mg; t = 4.394, P < 0.01] and fetal weights [(96.81 ± 11.63) mg vs. (122.86 ± 3.67) mg; t = 5.655, P < 0.01] were significantly lower in the WT infection group than in the WT control group, and the placental [(66.31 ± 9.00) mg vs. (83.13 ± 9.13) mg; t = 3.471, P < 0.01] and fetal weights [(74.32 ± 6.33) mg vs. (96.81 ± 11.63) mg; t = 4.495, P < 0.05] were significantly lower in the Tim-3^-/- infection group than in the WT infection group. No aborted fetuses were observed in the WT control group, and the abortion rate was significantly higher in the WT infection group [(30.14 ± 9.87)%] than that in the WT control group (t = 8.076, P < 0.01), while the abortion rate was higher in the Tim-3^-/- infection group [(66.85 ± 26.17)%] than in the WT infection group (t = 3.473, P < 0.01). Adverse pregnancy outcomes were significantly aggravated in pregnant mice in the Tim-3^-/- infection group than in the WT infection group. Flow cytometry measured lower percentages of Tim-3 positive cells in human dNK cells [(12.22 ± 4.61)% vs. (24.50 ± 7.10)%; t = 7.717, P < 0.05], dMϕ [(23.87 ± 5.26)% vs. (41.23 ± 3.84)%; t = 5.865, P < 0.05], dMDSCs [(10.07 ± 1.02)% vs. (17.53 ± 0.65)%; t = 7.798, P < 0.05] and dDCs [(11.01 ± 4.10)% vs. (18.53 ± 4.93)%; t = 9.592, P < 0.05] in the infection group than in the control group, and Western blotting assay determined lower relative Tim-3 expression in human dNK cells [(0.69 ± 0.09) vs. (1.03 ± 0.06); t = 7.562, P < 0.01], dMϕ [(0.74 ± 0.09) vs. (0.98 ± 0.06); t = 4.099, P < 0.05], dMDSCs [(0.79 ± 0.08) vs. (0.95 ± 0.02); t = 4.398, P < 0.05], and dDCs [(0.84 ± 0.04) vs. (0.97 ± 0.06); t = 5.455, P < 0.05] in the infection group than in the control group, and flow cytometry measured the largest reduction [(50.82 ± 5.92)%] in Tim-3 expression in dNK cells following T. gondii infections, with (42.37 ± 7.96)% and (42.08 ± 12.31)% reductions in dMDSCs and dMϕ, and the smallest decrease in dDC cells [(41.45 ± 7.64)%]. Like flow cytometric analysis, Western blotting assay determined a reduction in Tim-3 expression in all types of decidual immune cells following T. gondii infections, with the largest reduction in dNK cells [(33.06 ± 8.52)%], followed by in dMϕ [(22.84 ± 10.64)%], and mild reductions in dMDSCs [(16.25 ± 7.44)%] and dDCs [(13.6 ± 4.42)%]. Conclusion T. gondii infections in the first trimester significantly downregulates Tim-3 expression in major decidual immune cell subsets at the maternal-fetal interface, with the most pronounced effect on dNK cells, followed by dMϕ, dMDSCs, and dDCs. Downregulation of Tim-3 expression in various types of decidual immune cells following T. gondii infection may play an important role in the development of adverse pregnancy outcomes.