To establish a stable culture system of skin fibroblasts from Tree Shrew (Tupaia belangeri), and provide a primary cell model for related experiment on this specific animal species, the inner thigh skin was scraped to isolate primary cells using tissue mass adherence and collagenase Ⅰ digestion, respectively. The obtained cells were purified by using differential digestion with trypsin. MEM (with 10% FBS) and low Serum Growth Supplement added MEM (LSGS) were used for cell culture. The cells were identified by immunofluorescence and Western blot analysis, and the characteristics of cells′ survival and replication were further assessed. The results showed that collagenase digestion was more suitable for isolation of primary skin cells than method of tissue adherence (Fig. 1, 2). According to the survival and growth characteristics of isolated cells (Fig. 5, 6), the LSGS may provide a better culture condition for isolated cells in comparing with that of MEM (Fig. 4). Immunofluorescence observation (Fig. 8 and Fig. 9) and Western blot (Fig. 10) detection revealed that the isolated cells were primary skin fibroblasts. This effective method for isolation of primary skin fibroblasts from Tree Shrews was successfully established and their culture conditions were optimized for further investigation.