:5-Fluorouracil (5-FU) is one of the most well established chemotherapeutic agents in the treatment of esophageal cancer. Ribonucleotide reductase M1 (RRM-1) is the rate‑limiting enzyme in de novo DNA synthesis, and has been considered to play an important role in the 5-FU metabolic pathway. However, the means by which RRM-1 participates in the anticancer effects of 5-FU and cisplatin (CDDP) have not been well studied. Here, we show that RRM-1 significantly contributes to the induction of DNA damage by 5-FU in esophageal cancer cell lines. An assay of γ-H2AX focus formation, a marker of DNA damage, after 5-FU treatment revealed good correlation with the levels of RRM-1 protein expression. Moreover, the increased sensitivity and RAD51 focus formation induced by the combination treatment of 5-FU and CDDP were significantly repressed by RRM-1 depletion. These results suggest that RRM-1 is involved not only in the induction of DNA damage by 5-FU but also in the synergistic cytotoxic effect in the combination therapy of 5-FU and CDDP.