Objective To establish a stable orthotopic model with high green fluorescent protein (GFP) expression in nude mice and observe its biological features.Methods Human HCT116 colon cancer cells transfected with GFP pLPCX retroviral plasmid were used to build a subcutaneous tumor model in nude mice.Fifteen BALB/C nude mice were selected to underwent orthotopic transplantation of colon when the GFP-labeled tumor grew to 10 mm × 10 mm as observed by in vivo fluorescent microscopy.The growth and metastasis of orthotopically implanted colon cancer cells were observed with fluorescent imaging system at different time points.The differences of the tumor size measured by peripheral vernier caliper and fluorescent imaging system were analyzed using the t test,and the differences in different groups were analyzed using the analysis of variance.Results GFP-labeled colon cancer models were successfully established in all the 15 nude mice,and there was no surgery-related complications or death.Tumors marked by GFP were observed under fluoroscope in week 3.The size of the tumors progressively increased with time.The volumes of the orthotopically transplanted tumors obtained from global measurement using fluorescent imaging system were greater than those measured by peripheral vernier calipers at postoperative week 3,4,5,6,7,while no statistically significant difference was observed (t =-1.280,-1.115,-0.718,-0.199,-0.386,P ＞0.05).There was a significant difference in the interation of measure method and different time points (F =29.546,P ＜ 0.05).Eight nude mice survived at the end of the experiment,and tumor metastasis was observed in 6 mice.Conclusions It is technically feasible to construct GFP-labeled colon cancer orthotopic transplantation model.The mice model could be used for real-time,in vivo,non-invasive and dynamic observation and analysis of the growth and metastasis of tumor cells.