Objective To explore the proliferation characteristics of primary small intestinal epithelial cells of tree shrews and the characteristics of human rotavirus (RV) G1P[8] infection to these cells, and establish a model of tree shrew primary small intestinal epithelial cells infected with human rotavirus G1P[8]. Methods The primary small intestinal epithelial cells were obtained by collagenase XI and dispase I digestion from tree shrew. After purification and identification, the obtained primary small intestinal epithelial cells were infected with RV. Then, culture supernatants of infected cells were collected every 12 hours after infection. Viral titer and viral load were subsequently determined. Western blot and indirect immunofluorescence observation were used to detect the expression of RV protein VP6 in the primary cells. The infectivity of RV to the tree shrew primary cells was finally evaluated.Results After purification and identification of primary epithelial cells from the tree shrew, high purity above 90% primary tree shrew small intestinal epithelial cells was obtained. These primary small intestinal epithelial cells could be infected with RV virus by comparing the virus infectivity to primary renal cells, HCT116 cells and MA104 cells. The virus titer reached to 2.0×10⁵TCID₅₀/mL at 72 h after infection. Using Western blot and indirect immunofluorescence observation, the specific viral protein of VP6 was determined to be expressed in the tree shrew primary small intestinal epithelial cells, and were located in the cytoplasm from days 1 to 5. Conclusions The separation, purification and cultivation methods of tree shrew primary small intestinal epithelial cells are successful, and the tree shrew model of RV-infected the tree shrew primary small intestinal epithelial cells is successfully established.