Despite an initial response to EGFR tyrosine kinase inhibitors (EGFR‐TKI) in EGFR mutant lung cancer, most patients eventually become resistant and result in treatment failure. Recent studies have shown that epithelial to mesenchymal transition (EMT) is associated with drug resistance and cancer cell metastasis. Strong multiple gene signature data indicate that EMT acts as a determinant of insensitivity to EGFR‐TKI. However, the exact mechanism for the acquisition of the EMT phenotype in EGFR‐TKI resistant lung cancer cells remains unclear. In the present study, we showed that the expression of Notch‐1 was highly upregulated in gefitinib‐resistant PC9/AB2 lung cancer cells. Notch‐1 receptor intracellular domain (N1IC), the activated form of the Notch‐1 receptor, promoted the EMT phenotype in PC9 cells. Silencing of Notch‐1 using siRNA reversed the EMT phenotype and restored sensitivity to gefitinib in PC9/AB2 cells. Moreover, Notch‐1 reduction was also involved in inhibition of anoikis as well as colony‐formation activity of PC9/AB2 cells. Taken together, these results provide strong molecular evidence that gefitinib‐acquired resistance in lung cancer cells undergoing EMT occurs through activation of Notch‐1 signaling. Thus, inhibition of Notch‐1 can be a novel strategy for the reversal of the EMT phenotype thereby potentially increasing therapeutic drug sensitivity to lung cancer cells. J. Cell. Biochem. 113: 1501–1513, 2012. © 2011 Wiley Periodicals, Inc.