The aim of this study was to determine the antimetastatic efficacy of the combination of 5-fluorouracil (5-FU), leucovorin (LV), and irinotecan (CPT-11) (FOLFIRI) in an orthotopic nude mouse model of GFP-HCT-116 human colon cancer-expressing green fluorescent protein (GFP). The mice were randomly divided into four groups: Group 1: saline control; Group 2: 30 mg/kg 5-FU + 90 mg/kg LV; Group 3: 5-FU + LV + 16 mg/kg CPT-11 (FOLFIRI); and Group 4: 5-FU + LV + 24 mg/kg CPT-11. 5-FU and LV were administered on days 11, 16 and 21, and CPT-11 on days 12, 18 and 22. Survival in Groups 3 and 4 was significantly longer than that in Groups 1 and 2, although no dose-dependency on CPT-11 was observed. Analysis of the primary and metastatic tumors by GFP imaging, as well as that of oncogene expression in mesentery lymph nodes, demonstrated that tumor growth and metastasis were significantly inhibited or even prevented by FOLFIRI. Pathological evaluation also demonstrated that metastasis was also inhibited by FOLFIRI. The results of the present study suggest FOLFIRI prolongs survival by inhibiting metastasis.