Background: The aim of this study was to evaluate the efficacy and tolerability of a gemcitabine, 5-fluorouracil and leucovorin (GEMFUFOL) chemotherapy regimen as first line treatment of metastatic biliary tract cancer. Materials and Methods: All patients received folinic acid $400mg/m^2$ on day 1, 5-fluorouracil bolus $400mg/m^2$ on day 1, IV infusion of 5-fluorouracil $2400mg/m^2$ over 46 hours, and gemcitabine $1250mg/m^2$ on day 1. Results: A total of 29 patients with metastatic biliary tract cancer received GEMFUFOL regimen as the firstline treatment. The mean follow-up was 22.1 months (95%CI, 12.5-31.8). One patient (3.4%) achieved complete response, 5 (17.2%) had partial response, and 4 (13.8%) had stable disease. The median progression-free survival was 3.3 months (95%CI, 2.9-3.7), and the median overall survival was 8.8 months (95%CI, 3.5-14). The 1-year and 2-year survival rates were 58.6% and 30%, respectively. Grade 3 and 4 toxicity included neutropenia in 4 patients (13.7%), thrombocytopenia in 2 (6.8%), anemia2 (6.8%), and alopecia in 1 (3.4%). Two patients (6.8%) developed febrile neutropenia. A dose reduction was achieved in 8 patients (27.6%) while 5 patients had extended-interval dosage (17.2%) for toxicity. Conclusions: The GEMFUFOL chemotherapy regimen was generally efficacious and tolerable as a first-line treatment of metastatic biliary tract cancer.