Histone methylation is associated with various biological and pathological processes including cancer development. KDM6A is a candidate tumor suppressor gene that encodes a histone H3 lysine 27 (H3K27) demethylase. In this study, we discovered that ectopic expression of KDM6A antagonized TGF-β-induced epithelial-mesenchymal transition (EMT) and cell migration of lung cancer cell lines through its demethylase activity. KDM6A counteracted TGF-β-dependent changes in the expression of EMT-related genes such as CDH1/E-cadherin, FN1/Fibronectin, ZEB family and microRNA-200 family. Mechanistic investigations revealed that KDM6A inhibited the recruitment of EZH2 histone H3K27 methyltransferase and H3K27 methylation on the regulatory regions of the target genes such as CDH1 and microRNA-200 family. Knockdown of KDM6A did not proceed EMT by itself, but influenced the expression of specific target genes critical for EMT, suggesting that endogenous KDM6A was involved in EMT-inducing transcriptional program. This study demonstrated a novel regulatory role of KDM6A histone demethylase in the epigenetic control of EMT process in lung cancer cells.