OBJECTIVES: Oncogenic alterations of epidermal growth factor receptor (EGFR) signaling are frequently noted in non-small cell lung cancer (NSCLC). In recent decades, EGFR tyrosine kinase inhibitors (TKIs) have been developed, although the therapeutic efficacy of these inhibitor is restricted to EGFR-mutant patients. In this study, we investigated that clathrin-mediated EGFR endocytosis hampers the effects of gefitinib and sustains NSCLC cells with wild-type EGFR. MATERIALS AND METHODS: NSCLC cell lines (H358, Calu-3, SNU-1327, and H1703) were stimulated with the EGF and treated with gefitinib and endocytosis inhibitors (phenylarsine oxide (PAO) and Filipin III). Growth inhibition and apoptosis were evaluated. Immunofluorescence, immunoprecipitation, and western blot assay were performed to investigate EGFR endocytosis and determine the signaling pathway. Xenograft mouse models were used to verify the combination effect of gefitinib and PAO in vivo. RESULTS: We confirmed the differences in EGFR endocytosis according to gefitinib response in wild-type EGFR NSCLC cell lines. EGFR in gefitinib-sensitive and -refractory cell lines tended to internalize through distinct routes, caveolin-mediated endocytosis (CVE), and clathrin-mediated endocytosis (CME). Interestingly, while suppressing CME and CVE did not affect cell survival in sensitive cell lines significantly, CME inhibition combined with gefitinib treatment decreased cell survival and induced apoptosis in gefitinib-refractory cell lines. In addition, blocking CME in the refractory cell lines led to downregulate of p-STAT3 and inhibit nuclear localization of STAT3 in vivo, combination treatment with gefitinib and a CME inhibitor resulted in tumor regression accompanying apoptosis in xenograft mouse models. CONCLUSION: Clathrin-mediated EGFR endocytosis contribute primary resistance of gefitinib treatment and CME inhibition combined with gefitinib could be an option in treatment of wild-type EGFR NSCLC.