AIM: To investigate the effect of fetal bovine serum (FBS) on the proliferation and differentiation of murine corneal epithelial cells in vitro . METHODS: Mouse corneal epithelial cells (MCEs) were cultured in serum-free low-Ca²⁺ medium (KSFM) and KSFM supplemented with 100mL/L FBS, respectively. Population doublings (PDs) were determined. The expressions of corneal epithelial cell markers p63, keratin 19 (K19) and involucrin were investigated by RT-PCR and Western blotting analyses. RESULTS: Cells in KSFM were stably subcultured over 25 passages; however, none of the cell lines could pass P3 in KSFM with FBS. In KSFM, the cells showed typical cobblestone appearance and expressed p63, K19 and involucrin. After medium was supplemented with FBS, cells became homogeneous, large and squamous. Furthermore, both RT-PCR and Western blotting analyses showed that the expression of involucrin was increased significantly. CONCLUSION: FBS has effects of inhibiting proliferation and triggering differentiation of MCEs.