〔Abstract〕 To establish a method for the isolation, culture and identification of primary fibroblasts from salivary gland of neonatal rat. The salivary gland of neonatal SD rat was digested by 0.125% trypsin. The proliferation of fibroblast cells was detected by high content cell imaging system. Vimentin was detected by cell immunofluorescence and Western blot to identify fibroblasts. RT-PCR detected the expression level of vimentin mRNA, CD90 labeled fibroblasts to identify cell purity. The primary cells obtained from isolation were bright and spherical, with a clear outline, they were distributed separately or aggregated into cell clusters. Most of the cells were attached to the wall bottom after 90 min-culture; part of them began to stretch at this point. Cell monolayer was formed after 5 to 6 days. Cells presented as spindle-shaped or polygon-shaped after subculture. Positive vimentin expressing cells were identified as fibroblasts. The percentage of fibroblast reached to around 87% in passage 3. After 24 hours of culture in DMEM/F12 medium containing 5% FBS, the proliferation of cells could be detected. Trypsin digestion is an effective method for the isolation of fibroblasts from the salivary gland of neonatal rat.