5-Fluorouracil (5-FU) is a key drug for the treatment of esophageal squamous cell carcinoma (ESCC); however, resistance to it remains a critical limitation to its clinical use. To clarify the mechanisms of 5-FU resistance of ESCC, we originally established 5-FU-resistant ESCC cells, TE-5R, by step-wise treatment with continuously increasing concentrations of 5-FU. The half maximal inhibitory concentration of 5-FU showed that TE-5R cells were 15.6-fold more resistant to 5-FU in comparison with parental TE-5 cells. TE-5R cells showed regional copy number amplification of chromosome 1p including the DPYD gene, as well as high mRNA and protein expressions of dihydropyrimidine dehydrogenase (DPD), an enzyme involved in 5-FU degradation. 5-FU treatment resulted in a significant decrease of the intracellular 5-FU concentration and increase of the concentration of α-fluoro-ureidopropionic acid (FUPA), a metabolite of 5-FU, in TE-5R compared with TE-5 cells in vitro. Conversely, gimeracil, a DPD inhibitor, markedly increased the intracellular 5-FU concentration, decreased the intracellular FUPA concentration, and attenuated 5-FU resistance of TE-5R cells. These results indicate that 5-FU resistance of TE-5R cells is due to the rapid degradation of 5-FU by DPD overexpression. The investigation of 5-FU-resistant ESCC with DPYD gene copy number amplification and consequent DPD overexpression may generate novel biological evidence to explore strategies against ESCC with 5-FU resistance.