Although patients of non-small cell lung cancer (NSCLC) with somatic activating mutations of the epidermal growth factor receptor (EGFR) gene dramatically respond to the EGFR tyrosine kinase inhibitors (EGFR-TKIs) treatment, the majority of them eventually undergo disease progression. Cigarette smoke is the strongest documented risk factor for the development of lung cancer. Nicotine, while not carcinogenic by itself, has been shown to induce proliferation, angiogenesis, and epithelial-mesenchymal transition in several experimental models; these effects can be associated with resistance to EGFR-TKI. In this study, PC-9 and 11_18 cell lines (EGFR-mutated NSCLC cell lines) were cultured with 1 µM nicotine for 1 month (designated as PC-9/N and 11_18/N cell lines, respectively). In vitro, these cell lines were resistant to EGFR-TKI, whereas short exposure of nicotine (1 week) had no influence on the sensitivity. Phosphorylation of EGFR in PC-9/N and 11_18/N cell lines were less decreased than the controls and high concentration of EGFR-TKI could decrease the phosphorylation. Higher expressions of nicotinic acetylchorine receptor α1 subunit were found in PC-9/N and 11_18/N cell lines than the cell lines with short nicotine exposure or the controls. Finally, we investigated 72 EGFR-mutated NSCLC patients indicating that the progression-free survival (PFS) time of 24 smoker patients was significantly reduced compared to those with never-smoker (median PFS; 6.7 versus 14.4 months, respectively, p = 0.013). Taken together, our findings indicate that chronic exposure of nicotine by cigarette smoke mediates resistance to EGFR-TKI via EGFR signal. The resistance can be overcome by high dose EGFR-TKI.