The attainment of chemoresistance during tumor metastasis is often experienced. In this study, we evaluated the correlation between sensitivity to 5-fluorouracil (5-FU) and the mRNA expression level of several 5-FU-related metabolic enzymes [thymidylate synthase, dihydropyrimidine dehydrogenase (DPD), thymidylate phosphorylase (TP), orotate phosphoribosyl transferase, and uridine phosphorylase] in primary colorectal cancer and synchronous liver metastases from ten patients to investigate how colorectal cancer acquires 5-FU resistance during liver metastases. A liver metastasis model of xenotransplanted human colon cancer cell line (HCT116) in nude mice and several cell lines from metastatic liver tumors were also established and analyzed. Chemosensitivity and mRNA expression levels were measured by using collagen gel droplet-embedded culture drug sensitivity tests and real-time quantitative reverse transcription-polymerase chain reaction. Metastatic liver tumors were significantly more resistant to 5-FU than primary colorectal cancer (T/C, 88.7% versus 69.7%, p<0.05). DPD and TP mRNA levels were significantly higher in metastatic liver tumors (DPD: 10.36+/-1.81 versus 3.95+/-0.99, p<0.01; and TP: 18.80+/-4.96 versus 7.28+/-1.23, p<0.05) and inversely correlated with 5-FU sensitivity (DPD: R=0.570, p<0.05; TP: R=0.600, p<0.05). In the mouse model, metastatic liver tumors were significantly more resistant to 5-FU than HCT116 (T/C, 92.7%, 96.2% versus 68%, p<0.001). The DPD and TP mRNA levels increased with repeated liver metastases. DPD and TP may affect the acquisition of resistance to 5-FU during liver metastasis of colorectal cancer. This mouse model may be useful for analyzing the mechanisms of how colorectal cancer acquires resistance to 5-FU during liver metastases.