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Background: The optimal treatment strategy for biliary tract cancer(BTC) after curative?intent resection remains con?troversial. The purpose of this study was to evaluate the efficacy of fluoropyrimidine?based adjuvant chemotherapy for BTC patients undergoing microscopically margin?negative(R0) resection.Methods: We retrospectively analyzed the clinical data of BTC patients who underwent curative?intent R0 resection. Patients were eligible if they received either fluoropyrimidine?based adjuvant chemotherapy or observation after R0 resection.Results: A total of 153 patients were included. In the entire patient cohort, no significant differences were observed in 5?year overall survival(OS) rates(48.4% vs. 39.6%, P = 0.439) or 3?year recurrence?free survival(RFS) rates(49.1% vs. 39.5%, P = 0.299) between patients who received fluoropyrimidine?based adjuvant chemotherapy or observation. However, for patients with stages Ⅱ and Ⅲ BTC, chemotherapy significantly improved 5?year OS rate(52.4% vs. 35.6%, P = 0.002) and 3?year RFS rate(55.5% vs. 39.1%, P = 0.021) compared with observation.Conclusion: Fluoropyrimidine?based adjuvant chemotherapy may prolong the survival of patients with stages Ⅱ and Ⅲ BTC after R0 resection.