Objective: To compare the transcriptional activity of human telomerase reverse transcriptase (hTERT), carcino-embryonic antigen (CEA) and cytomegalovirus (CMV) promoters in human colon cancer LoVo and SW480 cells. Methods: After the primer sets were designed, the hTERT and CEA promoters were cloned by PCR amplification from the genome of colon cancer cells. The CMV promoter was removed from the original vector pLVX-EGFP-3FLAG by double digestion and PCR method, and the hTERT and CEA promoters were introduced into the vector to construct the recombinant plasmid pLVX-hTERTp-EGFP-3FLAG and pLVX-CEAp-EGFP-3FLAG. Colon cancer LoVo and SW480 cells were transiently transfected with the above two recombinant vectors and the original vector (containing CMV promoter) respectively, and the expressions of green fluorescent protein in the two cell lines were determined. Results: Results of PCR, enzyme digestion and sequencing showed that the cloning products and plasmid constructions were completely correct. The transcriptional activities (number of cells expressing green fluorescence/total number of cells) of CMV, hTERT and CEA promoters in LoVo cells were 54.7%, 33.0% and 9.5%, and in SW480 cells were 16.5%, 10.1% and 8.5%, respectively. All the differences had statistical significance (all P<0.05). Conclusion: In human colon cancer cells, the transcriptional activity of CMV promoter is the highest, hTERT promoter is second and CEA promoter is the lowest. These results may provide information for the study of targeted gene therapy of colon cancer.