Advanced nonsmall-cell lung cancer (NSCLC) patients with mutated epidermal growth factor receptor (EGFR) can remarkably benefit from target therapy of EGFR-tyrosine kinase inhibitors (TKIs). However, increasing drug sensitivity and improving outcomes of NSCLC patients to EGFR-TKI therapy remains a challenge. Several studies have shown a link between microRNAs and drug resistance in cancer. In this study, we hypothesized that the rs12740674 single nucleotide polymorphism in the enhancer of miR-1262 may affect its expression, which may impact the outcome of NSCLC patients treated with EGFR-TKIs. The rs12740674 polymorphism was genotyped in two independent cohorts, including 319 EGFR-TKI treated stage IIIB/IV NSCLC patients. The allele-specific regulation on miR-1262 transcription by rs12740674 and impacts of miR-1262 on gefitinib sensitivity were evaluated in vitro and in vivo. Cox regression analyses indicated that the rs12740674 T allele was significantly associated with short survival time in both cohorts (p < 0.05). Luciferase assays demonstrated that the rs12740674 T allelic enhancer showed weaker capability to promote miR-1262 transcription compared with the C allelic enhancer, which may be due to reduced transcription factor binding according to electrophoretic mobility shift assays. Furthermore, significantly decreased miR-1262 expression in NSCLC and nontumor lung tissues of T allele carriers was observed compared with levels in C allele carriers. Moreover, miR-1262 expression enhanced the anticancer effects of gefitinib on NSCLC cells. Our data indicate that miR-1262 may be a potential therapeutic target for NSCLC.