Objectives: The aim of this study was to examine the relevance of expression profiling of 4 genes involved in the action of gemcitabine among patients with pancreatic ductal-cell adenocarcinoma (PDAC). Methods: A group of 100 patients who underwent pancreatic resections for PDAC and received adjuvant chemotherapy with gemcitabine between 2007 and 2010 was identified. Expression of mRNAs for human equilibrative nucleoside transporter 1 (hENT1), ribonucleotide reductase subunits (RRM1, RRM2), and deoxycytidine kinase (dCK) was examined by quantitative real-time polymerase chain reaction, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and dichotomized into groups of low and moderate/high expression levels grouped by tertiles. Results: Significantly better median survival times were found for high/moderate expression levels of hENT1 (27.9 vs 12.4 months, P = 0.001) and dCK (19.7 vs 10.5 months, P = 0.003), as well as low expression of RRM1 (23.4 vs 11.4 months, P = 0.027). A Cox proportional hazards model identified low expression of hENT1 (hazard ratio [HR], 3.38; 95% confidence intervals [CI], 2.28-10.50) and dCK (HR, 2.24; 95% CI, 1.63-3.39), and high/moderate levels of RRM1 (HR, 1.65; 95% CI, 1.23-2.45) as negative prognostic factors. Conclusions: Expression of hENT, RRM1, and dCK genes provides important prognostic information for PDAC patients treated with adjuvant gemcitabine.