Objective To establish an enterovirus 71 (EV71) infection model of tree shrew primary renal cells. Methods Tree shrew primary renal cells were obtained by trypsin digestion. After subculture and purification, EV71 virus was used to infect these primary cells. The culture supernatant of these EV71-infected cells was collected for virus titer detection at 1, 2, 4, 6 and 8 days post-infection. The cells were collected for detection of EV71 VP1 protein by Western blot assay. Furthermore, the expression and location of VP1 protein in the infected cells were detected by indirect immunofluorescence assay. Vero cells were taken as positive control to evaluate the infectivity of EV71 virus to tree shrew primary renal cells. Results Morphologically, the cultured cells were proved to be majorly consisted of the primary renal cells after subculture and purification. The obtained primary cells were infected by EV71 virus. The virus titer was up to 1.3 × 10⁶ TCID₅₀/mL during 48-96 h post-infection, proving that EV71 virus infected and proliferated in the tree shrew primary renal cells. Western blot showed that the viral VP1 protein was detected from infected primary cells at 2 to 8 d post infection. VP1 protein was also observed in the cytoplasm at 2 to 6 d post infection by indirect immunofluorescence. Compared with Vero cells, the infectivity of EV71 virus to tree shrew primary renal cells and its proliferation were confirmed. Conclusions Based on the successful establishment of cell culture of tree shrew primary renal cells, the infectivity to the obtained cells and proliferation of EV71 virus in the cells are confirmed. The model of EV71 virus-infected tree shrew primary renal cells is initially established.