Objective To explore the role of cancer stem cells in EMT-induced acquired resistance to EGFR-TKIs in NSCLC. Methods The EGFR del E746-A750 mutated human lung adenocarcinoma PC-9 cell line and gefitinib acquired resistance cell line PC-9/AB were used in this study. EMT was assessed by western blotting assay. The sensitivity to gefitinib was tested with CCK8. Flow cytometry for antibody analysis was used to quantify CSCs within the cell lines. Results Compared with PC-9, PC-9/AB underwent EMT and showed no-table resistance to gefitinib (P < 0.01). Compared with PC-9, the proportions of CSCs were much higher in PC-9/AB. Conclusion EMT plays an important role in the acquired resistance to EGFR-TKIs in NSCLC , possibly through the up-regulation of CSCs.