Purpose <p>In previous studies, we discussed the effect of cilengitide, a cyclic Arg-Gly-Asp (RGD) peptide, and its derivatives on the growth of non-small cell lung cancer (NSCLC), TGF-β1-induced epithelial–mesenchymal transition (EMT), migration and invasion process. Cilengitide and some of its synthesized derivatives were found to have inhibitory effect on TGF-β1-induced EMT, migration and invasion of NSCLC when treated together with tyrosine kinase inhibitors (TKIs).</p> Methods <p>However, long-term administration of TKIs used for the treatment of NSCLC induces resistance in cells, so it is necessary to confirm the effect in NSCLC cells that are resistant to TKIs. In this study, we investigated the effects of cilengitide and its derivatives on afatinib-resistant NSCLC A549 (A549AR) cells.</p> Results <p>Cilengitide and its derivatives not only inhibited the migration and invasion of A549AR cells, but also showed a synergistic inhibitory effect when treated together with afatinib.</p> Conclusion <p>Although cilengitide and two derivatives have a somewhat minimal effect on inhibiting the growth of afatinib-resistant NSCLC cells, they have migration and invasion inhibitory effects, so they can be helpful in the treatment of metastatic carcinoma through co-administration with other types of anticancer drugs.</p>