Background: Cultivated oral mucosal epithelial cells (OMECs) are widely used in the treatment of limbal stem cell deficiency (LSCD) for their ocular reconstruction capability. As the most important component of the limbal microenvironment, limbal niche cells (LNCs) play a key role in the direction of stem cell differentiation. In this study, we investigated whether LNCs can induce the transdifferentiation of rat OMECs to corneal epithelial-like cells. Methods: We isolated OMECs and LNCs from rats by dispase and collagenase, respectively, to establish a three-dimensional or Transwell coculturing system. NIH-3T3 cells and renewed LNCs were also used as feeder layers in the Transwell system to compare their ability to support the OMECs. The airlift method was used for the culture of OMECs to obtain a stratified epithelial sheet. Cocultured OMECs were characterized by reverse-transcription polymerase chain reaction, Western blotting, hematoxylin and eosin staining, and immunohistochemistry. Results: The cocultured OMECs showed corneal epithelial-like morphology and expressed the corneal epithelial markers CK12 and Pax6 in most cocultured systems. Furthermore, we found that the expression level of CK12, Pax6, and proliferation marker Ki67 was upregulated when compared with that of other groups by renewing the LNCs in the Transwell system (p < 0.05, n = 3), suggesting that this might be a potential method for improving the efficiency of transdifferentiation. The obtained stratified epithelial sheet expressed CK3 and CK12. Conclusion: Through coculturing OMECs and LNCs in vitro, we successfully cultivated corneal epithelial-like OMECs. This investigation is of great significance for the treatment of LSCD and ocular surface reconstruction.