AIM: To investigate the effect of calcium on the proliferation and differentiation of murine corneal epithelial cells in vitro. METHODS: Mouse corneal epithelial cells were cultured in serum-free low-Ca2+ medium (KSFM) and KSFM supplemented with 0.9mmol/L Ca2+. Population doublings (PDs) were determined. The expression of corneal epithelial cell markers p63, keratin 19 (K19) and involucrin was investigated by RT-PCR analysis and semiquantitative analysis of Western blotting. RESULTS: Cells in KSFM were stably subcultured over 25 passages, however, none of the cell lines could pass P4 in KSFM with Ca2+. In KSFM, the cells was were homogeneous and small cells with typical cobblestone appearance; and expressed p63, K19 and involucrin. After medium was supplemented with calcium, cells became a heterogeneous mix of small and large cells. Furthermore, semiquantitative analysis of Western blotting showed that the expression of involucrin was increased significantly. CONCLUSION: Calcium has the effect of inhibiting pro- liferation and triggering differentiation on mouse corneal epithelial cells.