Abstract: Objective: Cytosolic 5'-nucleotidase (CN- Ⅱ), a nucleotide kinase, exhibits both 5'-nucleotidase and nucleoside phos-photransferase activities. Abnormal CN- expression may be correlated with the resistance of nucleoside analogs in anticancer drugs. This study was designed to investigate CN-Ⅱexpression in human non-small cell lung cancer (NSCLC) tissues and its correlation with the clinicopathological parameters as well as the prognosis of patients treated with gemcitabine. Methods:Immunohistochemistry was used to detect CN- Ⅱexpression in 116 cases of paraffin-embedded NSCLC samples. The correlations with the clinicopathological pa-rameters and the response to gemcitabine chemotherapy of CN- Ⅱwere analyzed through the Chi-square test. Log-rank test was used to determine whether or not CN-Ⅱexpression is correlated with the overall survival of patients. Results:The positive rate of CN- Ⅱwas 53.4% in 116 NSCLC tissues. No significant correlation existed between CN-Ⅱexpression and the clinicopathological parameters. Among the 67of the 116 patients who received gemcitabine chemotherapy, those with tumor progression (positive rate of 57.6%) exhib-ited higher CN- Ⅱexpression than those with therapeutic efficacy (positive rate of 30.4%,P=0.008) and disease-control chemotherapy (positive rate of 36.7% ,P=0.013). The progression-free survival was 4.5 and 5.5 months in the CN- Ⅱ-positive and CN-II-negative groups, respectively, with significant differences (95% CI:4.452 to 6.148, P=0.041). Correspondingly, the overall survival was 9.5 and 11 .0 months in the two groups (95% CI:8.667 to 13.333, P=0.282). Conclusion:CN- Ⅱmay be a prognostic factor for gemcitabine chemotherapy in NSCLC patients.