Background: Our previous randomized controlled trial, GEMSAP study (Br J Cancer. 2012 5;106:1934-9), showed a combination therapy of gemcitabine (Gem) & S-1 (GS) for advanced pancreatic cancer was superior to Gem monotherapy in terms of progression-free survival (PFS) but not overall survival (OS). Leucovorin (LV) is known to enhance efficacy of S-1 and we conducted this phase 1 trial of combination therapy of Gem, S-1 and LV (GSL). Methods: A primary endpoint of this classical “3+3” design phase 1 trial is to determine recommended dose of GSL. Inclusion criteria were 1. histologically-confirmed advanced pancreatic cancer without prior treatment, 2.PS 0-2, 3. age over 20. Treatment schedule was S-1 80 mg/m² 2x p.o. days 1-7, LV50mg 2x p.o. days 1-7, Gem 600(Level1), 800(Level2), 1000 mg/m²(Level3) div 30-min day 1 in a 2-week schedule. Dose-limiting toxicities were Grade 4 hematological and Grade ≥3 non-hematological toxicities, or delay of recovery from treatment-related toxicity for more than 2 weeks. Results: Between May 2012 and Feb 2013, 15 patients (Level 1/2/3: 6/6/3 patients) were enrolled; 7 males, a median age of 66, PS 0/1: 5/10, locally advanced/metastatic: 5/10. DLT was observed in 2/6 in Level 1 (Grade 3 anorexia in 1 and Grade 3 anorexia/stomatitis/diarrhea in 1) and 1/6 in Level 2 (Grade 3 pulmonary embolism). No DLT was observed in Level 3 and RD was determined as 1,000 mg/m²of Gem. Tumor response by RECIST was PR 5, SD 9, NE 1 with response rate of 33% and disease control rate of 93%. Overall toxicities greater than 3 were neutropenia 20%, anemia 7%, anorexia 13%, diarrhea 7%, stomatitis 7% and pneumonitis 7%. Conclusions: RD of GSL was determined as GEM 1000 mg/m² div 30 min day 1, S-1 80 mg/m² 2x, LV50mg 2x p.o. days 1-7. GSL was tolerable and showed promising tumor response in advanced pancreatic cancer. Clinical trial information: UMIN000007556.