BACKGROUND Epithelial-mesenchymal transition (EMT) induces the loss of cell-cell interactions in polarized epithelial cells and converts these cells to invasive mesenchymal-like cells. It is also involved in tissue fibrosis including that occurring in some ocular surface diseases such as pterygium and in subepithelial corneal fibrosis in limbal stem cell deficiency. Here, we examined the effects of the secretome of human adipose-derived mesenchymal stem cells (AdMSCs) on EMT in human corneal epithelial cells (CECs). METHODS EMT was induced with transforming growth factor-β (TGF-β) in primary human CECs isolated from the human corneal limbus. The effects of the AdMSC secretome on EMT in these cells or stratified CEC sheets were analyzed by co-cultivation experiments with the addition of AdMSC conditioned-medium. The expression of EMT-related genes and proteins in CECs was analyzed. The superstructure of CECs was observed by scanning electron microscopy. Furthermore, the barrier function of CEC sheets was analyzed by measuring transepithelial electrical resistance (TER). RESULTS The AdMSC secretome was found to suppress EMT-related gene expression and attenuate TGF-β-induced corneal epithelial dysfunction including the dissociation of cell-cell interactions and decreases in TER in constructed CEC sheets. CONCLUSIONS The secretome of AdMSCs can inhibit TGF-β-induced EMT in CECs. These findings suggest that this could be a useful source for the treatment for EMT-related ocular surface diseases.