The role of epithelial-to-mesenchymal transition in cancer drug resistance is increasingly acknowledged. We examined whether epithelial-to-mesenchymal transition affects gefitinib resistance in non-small cell lung cancer (NSCLC) cells. Cell viability was detected by CCK-8 assay, VIM expression levels were determined by quantitative real-time polymerase chain reaction. Western blot and immunocytochemistry were performed to determine the protein expression level of vimentin. We observed morphologic differences between gefitinib-sensitive and -insensitive cells. Compared with the sensitive parental cell line, HCC827, vimentin expression levels were increased in HCC827 cells with acquired gefitinib resistance. Vimentin expression was also markedly upregulated in cells with intrinsic gefitinib resistance, and upregulated vimentin expression was correlated with gefitinib sensitivity. Our previous study demonstrated that coadministration of gefitinib and GW3965 resulted in decreased cell proliferation and induced apoptosis. Therefore, we investigated the relationship among GW3965, vimentin, and gefitinib resistance in NSCLC cells by analysis of the expression of vimentin in cells treated with a combination of gefitinib and GW3965. Gefitinib treatment led to increased levels of intracellular vimentin, while combined treatment with gefitinib and GW3965 resulted in decreased vimentin expression levels through reduction of gefitinib drug resistance in NSCLC cells. Overall, these findings suggest that vimentin expression is associated with sensitivity to gefitinib, and our study highlights the potential usefulness of the drug, GW3965, for reversal of gefitinib resistance through inhibition of vimentin expression.