Bone marrow-derived mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into epithelial lineage, but the precise mechanisms controlling this process are unclear. Our aim is to explore the roles of Wnt/beta-catenin in the epithelial differentiation of MSCs. Using indirect co-culture of rat MSCs with rat airway epithelial cells (RTE), MSCs expressed several airway epithelial markers (cytokeratin 18, tight junction protein occudin, cystic fibrosis transmembrance regulator). The protein levels of some important members in Wnt/beta-catenin signaling were determined, suggested down-regulation of Wnt/beta-catenin with epithelial differentiation of MSCs. Furthermore, Wnt3alpha can inhibit the epithelial differentiation of MSCs. A loss of beta-catenin induced by Dickkopf-1 can enhance MSCs differentiation into epithelial cells. Lithium chloride transiently activated beta-catenin expression and subsequently decreased beta-catenin level and at last inhibited MSCs to differentiate into airway epithelium. Taken together, our study indicated that RTE cells can trigger epithelial differentiation of MSCs. Blocking Wnt/beta-catenin signaling may promote MSCs to differentiate towards airway epithelial cells.