Background: Patients with CCA usually present with advanced disease leading to the grave prognosis. Currently, cisplatin and gemcitabine is the standard treatment in advanced CCA. However, the CCA treatment outcomes are still poor and the options of treatment are quite limited. This study aimed to explore the efficacy and safety of S-1 plus leucovorin in patients with untreated advanced CCA. Methods: This single-arm two-center phase 2 study evaluated the efficacy and safety of S-1 40, 50 and 60 mg according to body surface area and leucovorin 15 mg , both given orally twice daily for one week, repeated every two weeks. Treatment was continued until complete 12 cycles, disease progression or unacceptable toxicity. The primary endpoints were overall response rate (ORR) and disease control rate (DCR) per RECIST version 1.1. The secondary endpoints were progression free survival (PFS), overall survival (OS) and toxicity. Results: Of total 32 patients and a median follow up time of 9.5 months, the ORR was 25% (95%CI 9.1-40.9) and the DCR was 62.5% (95% CI 44.8-80.2). In 25 response evaluable patients, the ORR was 32% (95% CI 12.4-51.7). The PFS was 8.0 (95%CI 5.59-10.4) months. The OS was 11.0 (95%CI 9.47-12.53). The most common grade 3 or 4 toxicities were anemia, mucositis and diarrhea. There was one patient discontinuing treatment due to treatment related toxicity. Conclusions: S-1 plus leucovorin was active and tolerable in patients with advanced CCA. Clinical trial information: TCTR20160313001.