Objective To establish the mouse infection model of Babesia microti Lishui isolate (isolated from one patient in Lishui, Zhejiang Province) and evaluate the change of parasitemia and pathological features during infection in the mice. Methods NOD-SCID mice were intraperitoneally inoculated with whole blood sample from a patient infected B. microti Lishui for species preservation and isolation. Fifty BALB/c mice were randomly assigned to infection group and control group, 25 mice in each group. The BALB/c mice in the infection group were intraperitoneally inoculated with 1.0 × 10⁷ infected erythrocytes from NOD-SCID mice and the BALB/c mice in the control group were inoculated with an equal volume of normal saline. The tail vein blood was collected daily to prepare the thin blood smear. The morphology of B. microti was observed under microscope after Giemsa staining, and the parasitemia was calculated. DNA was extracted from the infected mice samples. The 18S rRNA gene was amplified by Nest-PCR for sequencing, geneotyping, and phylogenetic tree analysis. At 0, 5, 10, 15, and 20 days post-infection, 5 mice in each group were selected to measure their body weight, spleen weight, and spleen length. The spleen tissue sections were prepared, and the pathological characteristics were observed under a microscope after HE staining. The routine blood test for samples from the infected mice was detected by an animal automatic blood analyzer. T-test was used for comparison between the two groups. Results At 5 days post-infection, the ring forms of the B. microti Lishui isolate were observed in the blood smear in the infection group. At 10 days post-infection, two or four parasites, in the shape of a double pear-shaped or Maltese cross, could be identified in the same erythrocyte, and hemolysis occurred. The parasitemia in mice in the infection group peaked (39.1 ± 4.6)% at 10 days post-infection and decreased to less than 1% after 20 days post-infection. The 18S rRNAs of the B. microti Lishui isolate and B. microti (GenBank accession number MT423326) share 98% sequence identity at the nucleotide level, and they also clustered in the same branch on the phylogenetic tree. At 10, 15 and 20 days post-infection, the body weight of mice in the infection group was (20.60 ± 1.02), (22.04 ± 0.77) and (22.78 ± 0.64) g, which was lower than that of the control group [(23.94 ± 0.84), (24.50 ± 0.26) and (24.64 ± 0.54) g] (t = 5.64, 6.78 and 4.99, P < 0.01). At 5, 10, 15 and 20 days post-infection, the spleen weight of mice in the infection group was (0.33 ± 0.02), (0.98 ± 0.11), (0.93 ± 0.04) and (0.67 ± 0.05) g, which was higher than that of the control group [(0.11 ± 0.01), (0.12 ± 0.01), (0.10 ± 0.02) and (0.11 ± 0.01) g] (t = 21.82, 22.25, 35.62 and 10.47, P < 0.01); the spleen length of the infected mice was (2.40 ± 0.12), (3.16 ± 0.06), (3.22 ± 0.05) and (2.98 ± 0.08) cm, which was higher than that of the control group [(1.76 ± 0.09), (1.74 ± 0.09), (1.74 ± 0.15) and (1.80 ± 0.07) cm] (t = 9.44, 30.27, 20.93 and 24.09, P < 0.01). At 10 days post-infection, splenomegaly, architectural distortion, blurring of the white pulp/red pulp border, massive lymphoproliferation, and congestion of splenic sinus were recognized in the spleen of mice in the infection group. The microanatomical structure of the spleen and the border region between the red and white pulp were recovered at 20 days post-infection. The rountin blood test results showed, at 10 days post-infection, the erythrocyte count, hematocrit, hemoglobin concentration, mean corpuscular volume, erythrocyte distribution width-standard deviation, erythrocyte distribution width-coefficient of variation, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration of mice in the infection group were (4.45 ± 0.32) × 10¹²/L, (27.72 ± 2.03)%, (86.2 ± 6.0) g/L, (60.7 ± 1.4) fL, (80.1 ± 4.0) fL, (31.9 ± 1.3)%, (19.4 ± 0.4) pg and (320.4 ± 3.8) g/L, respectively, which was higher than that of the control group [(9.55 ± 0.16) × 10¹²/L, (47.94 ± 1.64)%, (163.0 ± 4.8) g/L, (48.2 ± 1.1) fL, (27.7 ± 1.3) fL, (13.5 ± 0.5)%, (16.7 ± 0.7) pg and (339.0 ± 3.9) g/L] (t = 32.24, 17.34, 22.23, 15.71, 30.33, 28.41, 7.43 and 7.61, P < 0.01). At 10 days post-infection, the white blood cell count, monocyte count, monocyte percent, neutrophil count, and neutrophil percent of the mice in the infection group were (6.76 ± 0.87) × 10⁹/L, (0.78 ± 0.20) × 10⁹/L, (9.90 ± 0.87)%, (1.92 ± 0.42) × 10⁹/L and (27.74 ± 2.67)%, which was higher than that of the control group [(3.85 ± 0.26) × 10⁹/L, (0.17 ± 0.05) × 10⁹/L, (3.28 ± 0.40)%, (0.78 ± 0.15) × 10⁹/L and (21.20 ± 1.18)%] (t = 7.12, 6.54, 15.54, 5.71 and 5.00, P < 0.01). Conclusion A mouse infection model with B. microti isolated from patient in Zhejiang Lishui was established. After infection, the mice body weight was significantly reduced, accompanying with splenomegaly, spleen structural disorder and anemia.