Purpose: 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 III-IV 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). Conclusion: 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.