Background: The human equilibrating nucleoside transporter (hENT1) protein transports gemcitabine into the cell. However, role of hENT1 as a predictive biomarker for gemcitabine responsiveness in pancreatic adenocarcinoma (PDA) is still needed to be elucidated, although several studies had been conducted. We investigated whether hENT1 has a predictable role on adjuvant chemotherapy responsiveness in PDA according to stratification of hENT1 immunostaining. Methods: The 44 patients who underwent curative surgery (R0 or R1) due to PDA at Seoul National University Bundang Hospital from May 2015 to June 2017 were enrolled prospectively. The hENT1 expressions were measured by immunochemical staining (SP120-rabbit monoclonal antibody, Ventana). According to hENT1 immunostaing (≥ 50% vs. < 50%), higher hENT1 group received gemcitabine [1,000mg/m² (day 1, 8, 15) q 4 weeks x 6 cycles] and lower hENT1 group received [5-FU 425mg/m² (day 1-5) plus folinic acid 20 mg/m² (day 1) q 4 weeks x 6 cycles]. Primary outcome was overall survival (OS) and secondary outcomes were toxicity and recurrence free survival (RFS). Results: Among 44 patients, 5 patients were excluded due to withdrew consent (3 patients) and toxicities (2 patients in 5FU/LV group). Eighteen patients in the higher hENT1 group and twenty-six patients in the lower hENT1 group were followed up for 24.1 months. Our data showed OS was 97.4% at 12 months and 86.4% at 24 months. Moreover, RFS was 76.8% at 12 months and 64.2% at 24 months. Although it is preliminary data, it showed better OS and RFS compared to previous study (Neoptolemos JP et al., JAMA 2010) (OS, 61.3% at 12 months, 30.7% at 24 months; RFS, 58.7% at 12 months and 30.1% at 24 months). Conclusions: Adjuvant chemotherapy based on hENT1 immunostaining stratification provides excellent survival in resected PDA. Therefore, hENT1 immunostaining should be used for making a decision for adjuvant chemotherapy remimen in PDA. Clinical trial information: NCT02486497