Purpose. To evaluate the usefulness of the air-lifting technique for culturing corneal limbal epithelial cells on amniotic membrane (AM) for use in ocular surface reconstruction. A cultured sheet that has a good barrier function should be better for this purpose. In corneal epithelium, tight junctions (TJ) play a vital role in the barrier function. The TJ complex includes the integral transmembrane proteins occludin and the claudins, and some membrane-associated proteins such as ZO-1. In this paper, we investigated the barrier function and the expression of TJ related proteins. Methods. Corneal limbal epithelium obtained from donor corneas and cultivated on acellular AM was divided into two groups. These were the non-air-lifting (Non-AL) group, which was continuously submerged in medium, and the air-lifting (AL) group, which was submerged in medium for 3 weeks, then exposed to air by lowering the medium level. Morphology and the permeability to horseradish peroxidase (HRP) were determined by electron microscopy. Tight junction (TJ)-related protein and mRNA expression changes were assessed by immunoblotting and reverse transcription-polymerase chain reaction. Results. The cultures of both groups formed 4–5-layer-thick, well-stratified epithelium. The AL cultures had tightly packed epithelial cells with all the HRP/diaminobenzidine (DAB) reaction product accumulated on the apical surface of the superficial cells. The Non-AL culture, by contrast, had more loosely packed epithelial cells with larger intercellular spaces. The HRP/DAB reaction product penetrated the intercellular space to a depth of 3–4 cell layers. Statistically, there was a significant difference in intercellular spaces and desmosome count in the superficial cells between the groups. With AL, TJ-related proteins localized at the apical portion of the lateral membrane. TJ-related protein and mRNA amounts were not changed by AL while claudin subtype expression became more consistent and closer to that of in vivo corneal epithelium. Conclusions. The AL technique reduces intercellular spaces in the superficial cells and promotes the formation of the barrier function. It is useful in culturing corneal epithelial cells for use in ocular surface reconstruction.