Several polymorphisms in the genes involved in drug metabolism or elimination are known to be associated with pharmacokinetic or pharmacodynamic variability, but their correlations with overall survival (OS) and response rate (RR) in lung cancer have been unknown. This prospective study was to investigate whether genetic polymorphisms could influence OS and RR in advanced or metastatic non-small-cell lung cancer (NSCLC) patients treated with third-generation cytotoxic chemotherapy. Three hundred and sixty-four patients with chemotherapy naïve stage IIIB or IV NSCLC, receiving standard first-line chemotherapy, were eligible for this study. The DNA samples were extracted from peripheral blood mononuclear cells before treatment and single nucleotide polymorphisms of solute carrier family 29 member 3 SLC29A3 (rs10999776), SULT1E1 (rs4149525), and TBXAS1 (rs2267703), a category of drug-metabolizing enzymes or transporters were analyzed. Statistical analyses were performed by the log-rank test and Cox proportional hazards model. Patients with SLC29A3 C/T+T/T genotype had longer overall survival (median OS 12.3 months, 95 % CI 11.0-13.6 months) than those with C/C genotype (median OS 11.0 months, 95 % CI 9.9-12.1 months, P = 0.030 for log-rank test). More evidently significant association was found between the SLC29A3 polymorphism and overall survival in patients treated with gemcitabine-based chemotherapy (C/T+T/T versus C/C: median OS 12.0 months, 95 % CI 10.5-13.5 months versus median OS 10.0 months, 95 % CI 9.0-11.0 months, P = 0.027 for log-rank test). No association between the other two genetic polymorphisms and OS was observed. Genes involved in the drug metabolism or elimination (SLC29A3) may be new prognostic biomarkers for patients with advanced NSCLC who receive gemcitabine as the first-line chemotherapy and may unveil an unexplored molecular pathway correlated with the drug response and further may be predictive biomarkers for these patients.