Objective To understand the affects of Echinococcus multilocularismetacestode infection to natural killer T (NKT) cells and their subsets in mouse spleen. Methods Twenty-four C57BL/6 mice were randomly assigned into low, medium, high infection groups and control group (6 mice in each). The mice in low, medium, high infection groups were infected with 50, 500, 2 000 protoscoleces, respectively, through hepatic portal vein, while the mice in the control group injected with the same amount of saline. Two weeks after infection, the mice liver and spleen were collected to prepare liver tissue slices. The slices were stained with Masson routine method to observe the metacestodes lesion and liver fibrosis. Spleen lymphocyte suspensions were prepared for examining different NKT subsets, and the secreted cytokines by using flow cytometry.Results Two weeks after infection, there was no significant change in the mice liver of the control group. In the low and medium infection groups, only a small amount of granulomas were seen in the liver, together with weak inflammatory response which showed a conversion toward fibrosis repair; however, in the high infection group, a small germinal layer structure was formed in the liver, surrounded by dense inflammatory cells. The absolute numbers of splenic NKT cells in the medium and high infection groups were (6.32 ± 0.53) × 10 ⁵ and (7.37 ± 1.72) × 10⁵, respectively, which were higher than those in the control group and the low infection group [(5.02 ± 1.08) × 10 ⁵ and (4.90 ± 1.27) × 10 ⁵, P < 0.01]. The proportions of splenic NKT1-type cells secreting IFN-γ in the medium and high infection groups were (13.93 ± 1.61)% and (10.9 ± 3.04)%, respectively, which were lower than those in the control group and the low infection group [(17.3 ± 3.18)% and (19.42 ± 2.82)%, P < 0.01)]; the proportions of splenic NKT2-type cells secreting IL-4 in the low, medium and high infection groups were (5.62 ± 0.58)%, (5.86 ± 1.43)% and (5.92 ± 0.94)%, respectively, which were higher than that in the control group [(4.37 ± 0.83) %, P < 0.05)]; the proportion of splenic NKT17-type cells secreting IL-17A in the low and medium infection groups were (7.02 ± 1.19)% and (6.62 ± 0.41)%, respectively, which were higher than those in the control group and the high infection group [(4.68 ± 0.73)% and (3.73 ± 1.04)%, P < 0.01]. The absolute numbers of splenic CD4 ⁺ NKT cells in the medium and high infection groups were (1.75 ± 0.36) × 10 ⁵ and (1.82 ± 0.24) × 10 ⁵, respectively, which were higher than those in the control group [(1.24 ± 0.26) × 10 ⁵, P < 0.01]; the proportions of splenic CD4 ⁺ NKT1-type cells secreting IFN-γ in the low, medium- and high infection groups were (19.42 ± 2.82)%, (14.40 ± 1.81)%, and (12.5 ± 2.96)%, respectively, which were lower than those in the control group [(21.16 ± 2.83) %,P < 0.01]; the proportions of splenic CD4 ⁺ NKT17-type cells secreting IL-17A in the low, medium and high infection groups were (18.73 ± 1.99)%, (16.06 ± 1.42)% and (14.41 ± 3.55)%, respectively, which were higher than that in the control group [(10.27 ± 1.79)%,P < 0.01]. The proportions of splenic CD8 ⁺ NKT cells secreting IFN-γ in the low, medium and high infection groups were (10.04 ± 0.92)%, (13.43 ± 0.92)% and (9.24 ± 1.37)%, respectively, which were lower than those in the control group [(15.83 ± 1.59)%,P < 0.01]; the proportions of splenic CD8 ⁺ NKT cells secreting IL-10 in the medium and high infection groups were (4.07 ± 0.48)% and (3.92 ± 0.75)%, respectively, which were higher than that in the control group [(2.85 ± 0.64) %, P < 0.05]. The proportions of splenic DN NKT cells secreting IFN-γ in the medium and high infection groups were (17.48 ± 2.50)% and (14.06 ± 3.95)%, respectively, which were lower than that in the control group [(25.18 ± 5.78)%, P < 0.01]. Conclusion After infection of different numbers of E. multilocularismetacestode, the mice spleen NKT cells and their subsets changed significantly. The mice in the low and middle infection groups showed the immune response predominately with the CD4⁺ NKT17 type to kill and clear the parasites, while mice in the high infection group responded largely with the mixed subsets of CD4⁺ NKT17, NKT2 and NKT10 type cells, and NKT cells showed an inhibitory phenotype, leading to the decline of NKT1-type immune response, which is in favour of the parasite surviving.