Abstract: Objective：To isolate and culture the oral cancer associated fibroblasts (CAFs) and explore the possible role of CAFs in the invasion and metastasis of oral cancer cells in the tumor microenvironment. Methods：The tissue block culture method and enzymatic digestion method were used in the isolation, culture and purify of oral CAFs and normal mucous fibroblasts (NFs). And then the immunofluorescence assay and Western blot were used to identify the NFs and CAFs. Results：The oral CAFs were successfully isolated and cultured. They were long fusi form in shape, and the cells were in dense aggregations. The CAFs grew at certain point into multilayer structure and arranged in disorder. The cytokeratin 18 (CK18) was negative in NFs and CAFs, while the Vimentin was positive. The α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (FSP-1), fibroblast activation protein (FAP), and platelet derived growth factor receptor α(PDGFR-α) showed positive in CAFs compared to the NFs. The Desmin were expressed in NFs and CAFs. In the protein levels, the α-SMA, PDGFR-α were significantly increased in CAFs as compared to that in NFs. In addition, the FSP-1 was decreased in CAFs. However, there was no significant difference in the of Desmin and FAP in NFs and CAFs. Conclusions：In morphology, the oral CAFs and NFs were significantly different in growth and protein .