Phosphoribosylation of 5-fluorouracil (5-FU) is an essential step which leads to tumor growth inhibition and orotate phosphoribosyl transferase (OPRT) is the main enzyme that involves in this conversion of 5-FU to 5-fluorouridine monophosphate. This retrospective study was aimed to evaluate the correlation between tumor OPRT activity and the clinical outcome in colorectal cancer (CRC) patients treated by oral 5-FU-based adjuvant chemotherapy. Surgical specimen was obtained from resectable 124 CRC patients who were subsequently treated by oral 5-FU-based adjuvant chemotherapy. OPRT activity in the extract of tumor tissue was enzymatically determined. The cut-off value of intratumor OPRT activity against disease free survival was determined by maximal chi2 method. The disease free survival and overall survival in each group were calculated using the Kaplan-Meier method. Patients were divided into two groups by determined cut-off value of intratumor OPRT (0.147 nmol/min/mg protein) (high group: n = 102, low group: n = 22). Five-year DFS (P = 0.035) and OS (P = 0.020) were significantly better for high OPRT group. This study demonstrated that an assay of tumor OPRT contributes to the determination of 5-FU-based adjuvant chemotherapy outcome and application in clinical practice should be included in tumor analysis prior to 5-FU-based adjuvant chemotherapy.