Background: Acquired chemoresistance to 5-fluorouracil (5-FU) remains one of the obstacles for the success of 5-FU-based cancer chemotherapy, and some molecular mechanisms of acquired 5-FU resistance are still unknown. The main action of 5-FU is the suppression of DNA replication by inhibiting Thymidylate Synthase (TS). Materials and Methods: We analyzed 5-FU resistance mechanisms using the head and neck squamous cell carcinoma cell lines, UM-SCC-23, and two different resistant cell lines, UM-SCC-23/WR and UM-SCC-23/MR, which were procured from UM-SCC-23 cells. To acquire resistantance, the two cells underwent repeated treatment of 5-FU with different durations and frequency. We determined differences in the cell-cycle distribution and the expression of TS proteins in the three cell lines. Moreover, cell-cycle distribution in cells which acquired resistance after 5-FU treatment, was compared to that of parental cells, using flow cytometric analysis. Results: There was a remarkable increase in TS protein expression levels in UM-SCC-23/WR following 5-FU treatment. S-phase cells of UM-SCC-23 and UM-SCC-23/WR cells were immediately increased after treatment with 5-FU, whereas UM-SCC-23/MR were accumulated to the S-phase slightly later. Conclusion: The cell-cycle perturbation or elevation of TS protein expression may be involved in acquired 5-FU resistance and identifies 5-FU resistance mechanisms in the two different 5-FU treatment regimens.