Gefitinib, a selective EGFR tyrosine kinase inhibitor (EGFR-TKI), has been shown to block the signal transduction pathways implicated in the proliferation and survival of cancer cells. We previously demonstrated that the gefitinib-sensitive NSCLC cell line PC9 shows efficient ligand-induced endocytosis of phosphorylated EGFR (pEGFR). In contrast, the gefitinib-resistant NSCLC cell lines QG56 and A549 showed internalized pEGFR accumulation in the aggregated early endosomes, and this was associated with the sorting nexin 1 (SNX1), a protein that interacts with and enhances the degradation of EGFR upon EGF stimulation. To investigate the role of SNX1 on EGF-stimulated EGFR/pEGFR endocytosis via the endocytic pathway, we herein examined the effect of depletion of SNX1 expression by siRNA in human NSCLC cell lines. Using immunofluorescence, we demonstrated that transfection of SNX1 siRNA into gefitinib-resistant cells resulted in the disappearance of a large amounts of SNX1 staining. In addition, upon 15 min of EGF stimulation, we observed an efficient EGFR phosphorylation and a rapid endocytic delivery of pEGFR from early endosomes to late endosomes. Further, Western blot analysis revealed that silencing of SNX1 expression by siRNA in the gefitinib-resistant cells leads to an accelerated degradation of EGFR along with a dramatic increase in the amounts of pEGFR after EGF stimulation. Collectively, we suggest that SNX1 is involved in the negative regulation of ligand-induced EGFR phosphorylation and mediates pEGFR trafficking out of early endosomes for targeting to late endosomes in human lung cancer cells. These findings suggest that impairment of the protein trafficking machinery of SNX1 regulating pEGFR endocytosis might perturb pEGFR release from early endosomes, which subsequently leads to the acquisition of gefitinib-resistance in NSCLC cell lines.