Abstract: Pathogenic viruses can harm acutely the life and health of laboratory tree shrews acutely; however, few papers exist regarding natural pathogenic virus infection in this species. Six fecal samples obtained from dead tree shrews were collected. The fecal supernatant infected Vero cell line resulted in cytopathic effects (CPE) after 72 h. The CPE included granulating, shrinking, rounding, seining and falling off. Electron microscopy showed the isolation was spherical, double-layered capsid, and about 75 nm in diameter. The purified isolation genome was 10 segments in a typical 3:3:4 arrangements, as shown by polyacrylamide gel electrophoresis (PAGE). The isolation was confirmed by RT-PCR assays targeting the conserved region of the L1 gene, sequence analysis and reconstruction of a phylogenetic tree. The isolation was a Tupaia Orthoreovirus (TRV), belonging to Mammalian Orthoreovirus (MRV). The obtained strain had the closest phylogenetic relationship to the MRV strain T3/Bat/Germany/342/08. As a zoonotic virus, the novel TRV strain was first isolated from wild tree shrews, which is significant for promoting tree shrew standardization and providing scientific data for preventing zoonotic tree shrew-to-human transmission.