5-Fluorouracil (5-FU) is a cornerstone drug used to treat colorectal cancer (CRC). However, the prolonged exposure of CRC cells to 5-FU results in acquired resistance. We have previously demonstrated that levels of the 5-fluorodeoxyuridylate (FdUMP) covalent complex with thymidylate synthase (FdUMP-TS) and free-TS (native enzyme) are higher in 5-FU-resistant CRC cells than in the parental cell line (HCT116). Accordingly, resistant cells may have an efficient system for trapping and removing FdUMP-TS, thus imparting resistance. In this study, using a model of 5-FU-resistant CRC cells generated by repeated exposure, the role of autophagy in the elimination of FdUMP-TS in resistant cells was investigated. The resistant cells showed greater sensitivity to autophagy inhibitors than that of parental cells. Autophagy inhibition increased 5-FU cytotoxicity more substantially in resistant cells than in parental cells. Furthermore, autophagy inhibition increased FdUMP-TS protein accumulation in resistant cells. Our findings suggest that resistance to 5-FU is mediated by autophagy as a system to eliminate FdUMP-TS and may guide the use and optimization of combination therapies involving autophagy inhibitors.