Gemcitabine is widely used for the treatment of advanced biliary tract cancer (BTC) as first-line chemotherapy. However, there is no standard chemotherapy for patient with advanced BTC refractory to gemcitabine. We conducted a multicenter phase II study of S-1 monotherapy as second-line chemotherapy for patients with advanced BTC that were refractory to gemcitabine. S-1 was administered orally at a dose of 80 mg/m(2) for 28 days, followed by 14 days of rest. This regimen was repeated every 6 weeks. Tumor response was assessed every two cycles using the Response Evaluation Criteria in Solid Tumors version 1.0. Twenty-two patients were enrolled between March 2007 and January 2010, with 14 patients (64%) representing cases of recurrence after surgery. The overall response rate was 22.7%, and the overall disease control rate was 50.0%. The median overall survival time was 13.5 months (95% CI, 7.1-23.1 months) and the median time-to-progression was 5.4 months (95% CI, 2.6-17.2 months). Grade 3/4 toxicities included neutropenia (5%) and anemia (5%). The most common non-hematological toxicities were nausea (27%), anorexia (55%), and pigmentation (32%). In conclusion, S-1 monotherapy is feasible and moderately efficacious second-line chemotherapy for advanced BTC.