Thymidylate synthase (TS) is an enzyme responsible for DNA synthesis. Its competitive inhibition constitutes the major mechanism of the antitumor effect of 5-fluorouracil (5-FU) therapy, which significantly improves the survival rate of colon cancer patients. The aim of our study was to examine the clinical importance of TS expression in colon cancer patients and to correlate its expression with various clinicopathological parameters, tumor proliferative capacity, cell cycle-related molecules' expression and patients' survival. Of the 71 colon cancer patients studied, 51 (71.8%) tested positive for TS, with the positive result being statistically significantly correlated with patients' gender (P = 0.012), tumor histological grade (P = 0.032), vascular invasion (P = 0.017) and the expression of cyclin E, pRb and p16 (P = 0.042, P = 0.001 and P = 0.001, respectively). The overall 5-year survival rate was 40% for TS-positive patients and 68.6% for TS-negative ones (P = 0.0134); in patients aged >70 years, this was 30 and 77.8%, respectively (P = 0.0008). In a multivariate analysis of survival, TS expression proved to be of prognostic significance (P = 0.0174). Our findings support evidence for the clinical importance of TS expression in colon cancer patients and define it as an independent prognostic risk factor.