To improve the prognosis of cholangiocarcinoma, we investigated potential biomarkers that may enable the selection of patients for whom postoperative adjuvant chemotherapy is likely effective. The cohort of this retrospective study included 170 surgically resected cholangiocarcinoma patients, 26 with gemcitabine adjuvant chemotherapy (GEM group), 36 with S-1 adjuvant chemotherapy (S-1 group), and 103 receiving no adjuvant chemotherapy (NC group). Propensity score matching was performed to adjust patient backgrounds; 36 patients from the NC group then were selected. Immunohistochemistry of orotate phosphoribosyltransferase (OPRT) and human equilibrative nucleoside transporter 1 (hENT1) was performed to determine the correlation between their expression and disease-free survival (DFS). After matching, the backgrounds of these three groups were unbiased. No significant improvement of DFS by adjuvant chemotherapy was observed in the whole cohort. However, among the high-OPRT-expression patients, DFS of GEM, S-1, and NC groups at 5years was 28.8%, 53.8%, and 25.5%, respectively. The DFS of the S-1 group was significantly longer than that of the NC group (P=0.034). On the other hand, no significant differences in DFS were observed among the low OPRT expression patients. hENT1 expression was shown to have no predictive value. Multivariate analysis of the high-OPRT-expression patients demonstrated that S-1 adjuvant chemotherapy can reduce tumor recurrence (HR, 0.303; P=0.013). Cholangiocarcinoma patients with high OPRT expression would benefit from postoperative adjuvant S-1 therapy, which increases the DFS. Assessment of OPRT expression may contribute to the optimization of adjuvant chemotherapy for cholangiocarcinoma.