AIM: To investigate the effects of bovine pituitary extract on the proliferation of keratocytes and maintaining the keratocyte phenotype in vitro.METHODS: Single keratocytes were isolated by enzyme digestion for in vitro culture. Three groups were designed according to the different culture media:a bovine pituitary extract (BPE) group, a fetal bovine serum (FBS) group and the control group. The phenotypes and proliferation of cultured cells were evaluated by morphology, immunofluorescent staining and mRNA expression of CD34, Lumican, VSX1, α-SMA and proliferating cell nuclear antigen (PCNA). In the BPE group, cells underwent serial subcultivation, and their phenotypes were identified by immunofluorescent staining. To analyze the proliferation of keratocytesin differentconcentrations of BPE, six differentconcentrations were designed to ascertain the most appropriate amount.RESULTS: In the BPE group, the cells spread out and presented dendritic morphology, and their dendrites connected to one another to form networks. On the third passage, most cells maintained their phenotype. In the FBS group, the cells exhibited a dendritic appearance in early cultured stages, but their morphology subsequently changed into a fibroblast-like shape. The number of dendritic cells in BPE group was more than FBS and control groups. Immunofluorescent staining and real-time polymerase chain reaction (PCR) confirmed that few keratocytes underwent fibroblastic transformation in the BPE and control groups, and that proliferation was higher in the BPE group than in the control group. Although the proliferation was higher in the FBS group, many keratocytes underwent fibroblastic transformation.Theanalysis of cell morphology and mRNA expressions of CD34, PCNA and VSX1 in six group showed that different concentrations of BPE affected the proliferation obviously but didn’t affect the keratocyte phenotype, and the concentration of 40μg/mL was the most appropriate one.CONCLUSION: BPE can improve the proliferation of keratocytes and maintain their phenotype in vitro. Many keratocytes can be harvested rapidly and provide seeds for the construction of corneal stroma.