Background: The epithelial-mesenchymal transition (EMT) is a key process in tumor progression and metastasis and is also associated with drug resistance. Thus, controlling EMT status is a research of interest to conquer the malignant tumors. Materials and methods: A drug repositioning analysis of transcriptomic data from a public cell line database identified monensin, a widely used in veterinary medicine, as a candidate EMT inhibitor that suppresses the conversion of the EMT phenotype. Using TGF-beta-induced EMT cell line models, the effects of monensin on the EMT status and EMT-mediated drug resistance were assessed. Results: TGF-beta treatment induced EMT in non-small cell lung cancer (NSCLC) cell lines and the EGFR-mutant NSCLC cell lines with TGF-beta-induced EMT acquired resistance to EGFR-tyrosine kinase inhibitor. The addition of monensin effectively suppressed the TGF-beta-induced-EMT conversion, and restored the growth inhibition and the induction of apoptosis by the EGFR-tyrosine kinase inhibitor. Conclusion: Our data suggested that combined therapy with monensin might be a useful strategy for preventing EMT-mediated acquired drug resistance. (C) 2020 Elsevier Inc. All rights reserved.