Objective: To investigate the clinical efficacy of postoperative chemotherapy with tegafur/gimeracil/oteracil (S-1) in patients with cholangiocarcinoma. Methods: Eighty-seven patients with cholangiocarcinoma after radical resection were selected as study subjects. According to patients’ preferences, 42 cases received postoperative S-1 chemotherapy (chemotherapy group), and 45 cases did not receive any postoperative chemotherapy (non-chemotherapy group). The short-term effects and long-term results between the two groups of patients were compared. Results: The serum levels of KL-6 protein, CA19-9 and CA125 after chemotherapy were significantly reduced in chemotherapy group compared with those in non- chemotherapy group (all P<0.05). The main toxic and side effects during chemotherapy were dyspepsia (35.71%), anemia (14.29%) and pigmentation (14.29%), all of which were minor degrees of severity. The 1-year disease-free survival and overall survival rates showed no statistical difference between the two groups (both P>0.05), but the 3-year disease-free survival and overall survival rates in chemotherapy group were significantly higher than those in non-chemotherapy group (28.57% vs. 11.11%; 42.86% vs. 22.22%, both P<0.05). The median disease-free survival time and overall survival time was 13.7 and 21.5 months respectively, and both were significantly longer than those in non-chemotherapy group (9.8 and 16.4 months) (both P<0.05). Conclusion: In cholangiocarcinoma patients, postoperative S-1 chemotherapy has beneficial effects on reducing tumor marker levels and prolonging survival time.