Objective To investigate the biologic characteristic differences between gastric cancerassociated fibroblasts (GCAF) and human gastric mucosa fibroblasts (HGMF). Methods The fibroblasts were cultured primarily by outgrowth method and identified by immunohistochemistry. Third-generation cells were chosen to be analyzed. Their differences in biological features were compared by light microscopy, electron microscopy, and CCK-8 assay. The ability to synthesize proteins and cell cycle were detected by BCA Protein Assay Kit and flow cytometry respectively. Each experiment was repeated three times and the average was calculated. Results Compared with HGMF, the both of GCAF was larger and irregular with conjugate nuclei or multinucleate cells under the light microscopy. Deformed nuclei and multiple nuclei could be found in GCAF under the electron microscopy with elevated karyoplasmic ratio and increased mitochondria smooth endoplasmic reticulum. The ability of proliferation and protein synthesis in GCAF was significantly enhanced as compared with HGMF (P ＜0. 01 ), and both proliferation index (P ＜0. 05 ) and percentage of S phase cells ( P ＜ 0. 01 ) in GCAF were significantly higher than HGMF. Conclusion There were not only obviously differences in morphology, but also ability of proliferation, protein synthesis and cell cycle activity in GCAF were significantly higher than HGMF.