MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of gene expression, involved in diverse physiological and pathological processes. An oncogenic or tumor-suppressive miRNA may have potential as a therapeutic target to control cancers. Gefitinib is a tyrosine kinase inhibitor that targets epidermal growth factor receptor (EGFR). H460 and A549 cells with EGFR receptor-independent over-activation of protein kinase B (Akt) or extracellular signal-regulated kinases (ERK) are significantly resistant to gefitinib. The first aim of this study was to confirm a role for three miRNAs (let-7a, hsa-miR-126, and hsa-miR-145) in the inhibition of proliferation in non-small cell lung cancer (NSCLC) cells. A second aim was to evaluate three miRNAs for their abilities to overcome cellular resistance and enhance the gefitinib cytotoxicity. The expression of miRNAs was estimated by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation was examined by sulforhodamine B assay and tumor xenografts were measured in SCID/beige mice. The activation of Akt and ERK was observed by Western blotting. Forced expression of individual miRNA suppressed the growth of two cell lines and xenografts. The effect varied among different miRNAs and cells. Restoration of hsa-miR-126 more obviously inhibited cell growth than did restoration of hsa-miR-145 in both cells, and the suppressive effect was more significant in H460 xenografts than in A549 xenografts. Western blotting revealed that the inhibition of cell proliferation resulted from the inhibition of the activation of Akt and ERK. Moreover, forced expression of miRNAs contributed to enhanced cytotoxicity induced by gefitinib in lung cancer cells; especially in hsa-miR-126, the highest value of half max inhibitory (IC50) was increased sixfold. These findings confirm that tumor-suppressive miRNAs can inhibit the growth of NSCLC cells and enhance the targeted agents cytotoxicity, suggesting novel potential approaches to an improvement in chemotherapy.