Background: Cisplatin plus gemcitabine (GC) is considered as standard first-line chemotherapy for patients with advanced biliary tract cancer (BTC). However, no standard therapy is established for second-line setting. Some clinical studies showed that gemcitabine was more effective if administered by the fixed dose rate method, and S-1 had anti-tumor effect as second-line chemotherapy for BTC. We evaluated the efficacy and safety of combination therapy of fixed dose-rate gemcitabine and S-1 after failure of GC or gemcitabine alone. Methods: This study was a single arm phase II study, and primary endpoint was response rate (RR) with MinMax two stage design to test the hypothesis that RR was more than 7%. Number of patients needed was 35 with a power of 90% and two-sided α value of 0.05. Patients received gemcitabine (1,000 mg/m², div, over 100 minutes, day 1) and S-1 (40 mg/m² twice daily, oral, day 1-7), every two weeks until disease progression or intolerable adverse events. Results: Forty patients were enrolled from July, 2011 to Nov., 2014. Of 23 patients in the first stage, two patients showed response and proceeded to the second stage. Overall response rate was 7.5%. Overall survival, progression-free survival and disease control rate were 7.7 months, 2.6 months, and 47.5%, respectively. Common adverse events were anemia (97.2%), thrombocytopenia (66.7%), leukopenia (47.2%), neutropenia (41.7%), anorexia (39.0%) and fatigue (37.8%). Grade 3–4 adverse events were leukopenia (19.5%), neutropenia (19.5%), anemia (14.6%), thrombocytopenia (7.3%) and anorexia (4.8%). Biliary tract infection led to death in two patients; however, these were reported as not related to this study treatment. Conclusions: Fixed dose-rate gemcitabine plus S-1 was tolerable as second-line settings in BTC; however, its activity was modest for patients refractory to the standard gemcitabine. Clinical trial information: UMIN000005918 Clinical trial information: UMIN000005918.