OBJECTIVE: Human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) are a kind of multipotent cells with strong differentiation potential in vitro. With characteristics of low immunogenicity, hUC-MSCs have attracted much attention. This study aims to explore the feasibility of hUC-MSCs differentiating into corneal epithelial-like cells. STUDY DESIGN: hUC-MSCs were cultured in the conditioned medium from rabbit corneal epithelial cells. The induced hUC-MSCs were identified by immunofluorescence staining. The hUC-MSCs were inoculated on corneal stromal laminas and attached to the denuded posterior surface of the rabbit corneas. Rabbits received laminas with and without hUC-MSCs (hUC-MSCs and control group). Hematoxylin-eosin and immunofluorescence staining with transmission electron microscopy were carried out to evaluate corneal damage. RESULTS: The hUC-MSCs were morphologically spindle shaped, and rabbit corneal epithelial cells were cobblestone-like shaped. Cultivated hUC-MSCs were positive for CD44, CD29, and CD105 but negative for CD45 and CD34. The induced hUC-MSCs expressed CK3/12, CK14, CK19, and PNCA with corneal epithelial cell characteristics. Importantly, corneal transparency gradually recovered in the hUC-MSC group with decreased corneal opacity and neovascularization. CONCLUSION: Collectively, hUC-MSCs have the potential to differentiate into corneal epithelial cells, providing an accessibly alternative therapeutic source of stem cells for replacement of the corneal epithelium by tissue engineering technology. © Science Printers and Publishers, Inc.