Patients with non-small cell lung cancer (NSCLC) who have activating epidermal growth factor receptor (EGFR) mutations derive clinical benefit from treatment with EGFR-tyrosine kinase inhibitors ((EGFR-TKIs)- namely gefitinib and erlotinib. However, these patients eventually develop resistance to EGFR-TKIs. Despite the fact that this acquired resistance may be the result of a secondary mutation in the EGFR gene, such as T790M or amplification of the MET proto-oncogene, there are other mechanisms which need to be explored. MicroRNAs (miRs) are a class of small non-coding RNAs that play pivotal roles in tumorigenesis, tumor progression and chemo-resistance. In this study, we firstly successfully established a gefitinib resistant cell line-HCC827/GR, by exposing normal HCC827 cells (an NSCLC cell line with a 746E-750A in-frame deletion of EGFR gene) to increasing concentrations of gefitinib. Then, we found that miR-214 was significantly up-regulated in HCC827/ GR. We also showed that miR-214 and PTEN were inversely expressed in HCC827/GR. Knockdown of miR-214 altered the expression of PTEN and p-AKT and re-sensitized HCC827/GR to gefitinib. Taken together, miR-214 may regulate the acquired resistance to gefitinib in HCC827 via PTEN/AKT signaling pathway. Suppression of miR-214 may thus reverse the acquired resistance to EGFR-TKIs therapy.