Objective To explore the role and possible mechanism of grainyhead-like protein 2 (GRHL2) down-regulation in acquired drug resistance to tumor-targeted therapeutic drug gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI). Methods Human colon cancer cell line DiFi and human lung adenocarcinoma cell line HCC4006 were cultured in a stepwise increasing concentration of gefitinib to obtain gefitinib-resistant cell lines. The differentially expressed genes between gefitinib-resistant cell lines and parent cells were selected by RNA sequencing and verified by real-time fluorescent quantitative PCR (qRT-PCR). The pcDNA3.1-GRHL2 plasmid was transfected into the gefitinib-resistant cell lines to overexpress GRHL2, and the sensitivity of the cells to gefitinib was detected by CCK-8 method. The expression of epithelial marker (E-cadherin) and mesenchymal marker (Vimentin) in the gefitinib-resistant cells was detected by Western blotting. The relationship between GRHL2 expression and expression of E-cadherin and Vimentin in 60 human tumor cell lines was analyzed by CellMiner^TM database. Results We successfully obtained two gefitinib-resistant cell lines. RNA sequencing and qRT-PCR confirmed that the expression of GRHL2 in the gefitinib-resistant cells was decreased, while the sensitivity of the cells to gefitinib was restored after overexpressing GRHL2 in the gefitinib-resistant cells. Western blotting analysis showed that the E-cadherin expression was decreased and the Vimentin expression was increased in the gefitinib-resistant cell line. CellMiner^TM database analysis showed that the expression of GRHL2 was highly consistent with the ratio of E-cadherin to Vimentin. Conclusion Down-regulation of GRHL2 mediates drug resistance of tumor cell to gefitinib by inducing epithelial-mesenchymal transition.