Gemcitabine plus cisplatin (GC) is the standard treatment of advanced biliary tract cancer (BTC); however, it causes nausea, vomiting, and anorexia, and requires hydration. Gemcitabine plus S-1 (GS) reportedly has equal to, or better, efficacy and an acceptable toxicity profile. We aimed to confirm the non-inferiority of GS to GC for patients with advanced/recurrent BTC in terms of overall survival (OS). We undertook a phase III randomized trial in 33 institutions in Japan. Eligibility criteria included chemotherapy-naïve patients with recurrent or unresectable BTC, an Eastern Cooperative Oncology Group Performance Status of 0−1, and adequate organ function. The calculated sample size was 350 with a one-sided α of 5%, a power of 80%, and non-inferiority margin hazard ratio (HR) of 1.155. The primary end point was OS, while the secondary end points included progression-free survival (PFS), response rate (RR), adverse events (AEs), and clinically significant AEs defined as grade ≥2 fatigue, anorexia, nausea, vomiting, oral mucositis, or diarrhea. Between May 2013 and March 2016, 354 patients were enrolled. GS was found to be non-inferior to GC median OS: 13.4months with GC and 15.1months with GS, HR, 0.945; 90% confidence interval (CI), 0.78–1.15; P=0.046 for non-inferiority. The median PFS was 5.8months with GC and 6.8months with GS (HR 0.86; 95% CI 0.70–1.07). The RR was 32.4% with GC and 29.8% with GS. Both treatments were generally well-tolerated. Clinically significant AEs were observed in 35.1% of patients in the GC arm and 29.9% in the GS arm. GS, which does not require hydration, should be considered a new, convenient standard of care option for patients with advanced/recurrent BTC. This trial has been registered with the UMIN Clinical Trials Registry (http://www.umin.ac.jp/ctr/index.htm), number UMIN000010667.