In this study, we investigated the activity of single agent gemcitabine in the second-line setting of non-small cell lung cancer (NSCLC). File records of 21 patients treated with single agent gemcitabine in advanced NSCLC who received one prior chemotherapy including a taxane and platinum combination were retrospectively evaluated. Treatment consisted of IV gemcitabine 1,250 mg/m2on days 1 and 8, followed by a 1-week rest repeated every 3 weeks. A partial response was achieved in four (19%) patients. The median response duration was 16 (range, 12–32) weeks. Six (29%) patients had a SD more than 3 months. The median time to progression was 16 (range, 8–32) weeks. No complete response was observed. Median overall survival was 36 weeks for second-line gemcitabine in all patients (95%: CI 5–13 months). Hematological toxicity (all grades) was reported by 9 (42.9%) patients. One (4.75%) patient experienced grade 3/4 neutropenia. Grade 3/4 nausea and vomiting and mucositis were reported in one (4.75%) patient. In conclusion, this study shows that single agent gemcitabine is active and well tolerated as a second-line therapy for advanced NSCLC.