Objective: To explore the therapeutic efficacy and safety of chemotherapy sequential epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) for advanced non-small cell lung cancer (NSCLC) patients with EGFR-TKI acquired resistance. Methods: The clinical features of 96 advanced NSCLC patients with EGFR mutation positive and EGFR-TKI acquired resistance from January 2013 to December 2016 were retrospectively reviewed. Forty-five patients who received chemotherapy sequential EGFR-TKI were enrolled in observation group, and another fifty-one who accepted chemotherapy alone were enrolled in control group. The objective response rates, disease control rates and adverse effects were compared between two groups. Results: Compared with that of the control group, the objective response rate of the observation group was significantly higher [24.4% (11/45) vs.11.8% (6/51), P=0.037], the disease control rate was also significantly increased [77.8% (35/45) vs.52.9% (27/51), P=0.023], the progression free survival was obviously prolonged [(7.4 ± 2.0) months vs.(4.5 ± 1.2) months, P=0.029], the incidence of rash in adverse reactions was significantly increased [35.6% (16/45) vs. 7.8%(4/51), P=0.008]. There were no significant difference in the side effects between the two groups (P > 0.05). Conclusions Compared with chemotherapy alone, chemotherapy combined with sequential EGFR-TKI could bring better clinical efficacy for NSCLC patients with EGFR-TKI acquired resistance, and the adverse effects could be tolerated.