Background: Biliary tract cancer (BTC) is a highly lethal disease. The prognosis remains poor even after macroscopically curative resection due to the high recurrence rate. Therefore, developing effective adjuvant therapy is essential to improve treatment outcomes. In 2021, a phase III trial (JCOG1202: ASCOT) showed the superiority of adjuvant S-1 chemotherapy to surgery alone in the overall survival (OS) in BTC patients with curative resection (hazard ratio [HR] 0.694, 95% confidence interval [CI]: 0.514–0.935; p=0.008). Consequently, adjuvant S-1 chemotherapy has become the standard of care for resected BTC. In contrast, only a few studies have been conducted to assess the effectiveness of neoadjuvant therapy for BTC until now. Recently, a phase III trial (KHBO1401-MITSUBA) demonstrated the superiority of gemcitabine + cisplatin + S-1 (GCS) therapy to gemcitabine + cisplatin in patients with advanced unresectable BTC in terms of OS (HR 0.79, 90% CI: 0.628–0.996; p=0.046). The GCS arm showed a high response rate (41.5%), with promise in a neoadjuvant setting. This phase III trial aims to confirm the superiority of neoadjuvant GCS to surgery first in patients with resectable BTC. Methods: The main eligibility criteria are: (1) histologically diagnosed with adenocarcinoma or adenosquamous carcinoma; (2) diagnosed as resectable BTC with clinical stage II–IVA for perihilar bile duct cancer, stage IB–III for distal bile duct cancer, stage IIIA–IVA for gallbladder cancer, stage IIA–III for ampullary cancer, or stage III–IVA/stage IVB (T4N1M0) for intrahepatic bile duct cancer by diagnostic imaging; (3) Eastern Cooperative Oncology Group Performance Status 0 or 1; and (4) age ≥20 years. Enrolled patients are randomized 1:1 to neoadjuvant GCS + surgery + adjuvant S-1 or surgery + adjuvant S-1 regarding the center and primary site (perihilar/distal/gall bladder/ampulla of Vater/intrahepatic) by the minimization method. The GCS regimen comprises gemcitabine (1000 mg/m²) and cisplatin (25 mg/m²) on day 1 and S-1 (80-120 mg/day) on days 1–7, every 2 weeks for 3 courses. After surgery, S-1 (80-120 mg/day) for 4 weeks-on, 2 weeks-off, for a total of 4 cycles, is administered in both arms. The primary endpoint is OS, and secondary endpoints are progression-free survival (PFS), OS and PFS in patients who underwent R0/R1 resection, incidence of non-resection, %R0 resection, %R0/R1 resection, %lymph node metastasis, postoperative complications of Clavien–Dindo Grade IIIa or severer, serious adverse events, and objective response rate in the GCS arm. We calculated a sample size of 330 patients to obtain 70% power at a one-sided alpha error of 5%, a hazard ratio of 0.698 (3-year OS 67% vs 75.6%), an accrual period of 5 years, and a follow-up period of 3 years. The patient accrual was started in March 2021, and 126 patients have been enrolled as of September 2022. Clinical trial information: jRCTs031200388.