5-Fluorouracil (FU) is a chemotherapeutic agent commonly used against esophageal cancer. Recently, interferons (IFNs) have been administered together with cytotoxic chemotherapy to patients with this cancer, although the mechanisms of synergy are unknown. We reconsidered the mechanisms for the effects of 5-FU in this context, aiming to refine combination therapy. After three cell lines (T.T, TE-2, and TE-6), derived from human esophageal squamous cell carcinoma (SCC), were exposed to 5-FU, the expression profiles were analyzed using high-density oligonucleotide microarrays representing about 12,000 genes. Among them, three IFN-related genes, an IFN receptor gene (IFNAR2) and two IFN-stimulated genes (ISG15K, ISG-54K), that were up-regulated following addition of 5-FU, were investigated. Up-regulation was confirmed by RT-PCR. Based on these results, the antitumor effects of exposure to 5-FU simultaneously with IFN-α, β and -γ were investigated. The growth of esophageal SCC cells with 5-FU was suppressed synergistically or semi-additively by IFN-α and β, but not by IFN-γ. These findings indicated that 5-FU stimulated IFN pathways in esophageal SCC cells following up-regulation of the IFN type I receptor. A combination of 5-FU and an IFN, therefore, may be particularly efficacious in esophageal cancer.