Objective: To explore the impact of V5-epitope tag inserted in the commercial pcDNA5/FRT/V5-His TOPO expression vector on the metabolic activation of AFB1 by human CYP2A13. Methods: A C-terminal 6×Histag was first introduced into CYP2A13 cDNA by PCR and subsequently transferred into the expressing vector pcDNA5/FRT. Another commercial pcDNA5/FRT/V5-His TOPO expression vector was used to develop the construct directly via PCR. Both of the constructs were then transfected into Flp-In CHO and allowed for the stable expression of CYP2A13. The mouse CYP2A5 and the vector alone were used as positive and negative control, respectively. The presence of CYP2A5 and CYP2A13 cDNA and their protein expression in the stable transfectant cells were determined by immunoblotting assay using a monoclonal antibody against 6×Histag. The AFB1-induced cytotoxicity in these tranfected CHO cells were conducted by MTS assay and the IC50 of cell viability was used to compare the CYP enzyme metabolic activity in AFB1 metabolism among these cells. Results: In accordance with the Flp-In system working mechanism, all the transfectant cells presented same protein expression level. The CHO cells expressing CYP2A5 was more sensitive to AFB1 treatment than those cells expressing CYP2A13, there was about 30-fold IC50 difference between the two cells (2.1 nmol/L vs 58 nmol/L). Interestingly, CYP2A13 fused with V5-Histag had the lost of metabolic activity to AFB1 than that fused with Histag alone, the IC50 of the viability in CHO-2A13-His-V5 cells was about 20-fold less than CHO-2A13-His (＞1 000 nmol/L vs 58 nmol/L). However, there was no change between CYP2A5 fused with V5-Histag and Histag alone (2.4 nmol/L vs 2.1 nmol/L). Conclusion: The results demonstrate that CYP2A13 fused with V5-epitope has a significant impact on its metabolic activation to AFB1, which indicated that it should be careful to select a new expressing vector for evaluating the enzyme activity in carcinogen metabolism.