OBJECTIVE With the improvements in first- and second-line treatments in non-small cell lung cancer (NSCLC), there is an increasing number of patients who receive third-line therapy. No other standard choice for third-line therapy aside from erlotinib is possible. This study investigated the efficacy and safety of single-agent chemotherapy, epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), doublet chemotherapy and chemo-targeted therapy as third-line treatment in advanced NSCLC. METHODS This study included 233 stage IIIb or IV NSCLC patients who were retrospectively reviewed to explore the differences in survival between different treatments. RESULTS The median progression free survival (PFS) in the EGFR-TKIs, single-agent, doublet and chemo-targeted groups was 3.83, 2.72, 2.86 and 3.29 months, respectively (p = 0.073). The median OS from the initiation of the third-line treatment was 11.16, 8.24, 8.49 and 9.33 months in the 4 groups (p=0.02). The rates of grade IIIIV toxicities were 16.4, 27.6, 57.3 and 44.0% ( p <0.001), respectively with the third-line treatment, and overall survival (OS) was prolonged in patients who never smoked (p=0.040), had adenocarcinoma (p=0.034), had good ECOG performance status (PS) (p=0.012) and achieved disease control after both first-and second-line treatments (p =0.031). CONCLUSIONS Patients with advanced NSCLC who never smoked, had adenocarcinoma, have good PS, and good disease control from the first- and second-line therapies could benefit more with third-line treatment. EGFR-TKIs and chemo-targeted therapy showed increased OS compared with single-agent and doublet chemotherapy.