Abstract: Objective To identify the RNA interference action of recombined pSUPER-NRF2 vectors for the expression of NRF2 gene in colon cancer cells. Methods Two sequences targeting the ORF of NRF2 were cloned into the RNA polymerase III based expression vector pSUPER. These recombinants were transfected into Caco-2 cells. Fluorescence microscopic examination and Flow cytometry were performed after being transfected with pEGFP-N1 plasmids to observe the lipfectin transfection efficiency. The stable cells were selected in medium 48h after co-transfected pEGFP-N1 with G418. The expression of NRF2 was assayed using RT-PCR and Western blot. Results The construction of the recombinant expression vector pSUPER-NRF2-A1、B1 and its control vector pSUPER-NRF2-A2、B2 were successfully confirmed by the results of enzyme digestion, electrophoresis and sequencing. The transfection efficiency were 45.6％、74.3％、53.0％ and 46.5％ respectively in 24、48、72 and 96h。We compared the ability of these vectors to inhibit NRF2 in a transient and stable expression experiment. Importantly, pSUPER- NRF2-B1 was able to significantly knockdown NRF2 expression. pSUPER- NRF2-A1 only had a moderate activity, whereas pSUPER- NRF2-A2、B2 were inactive in this assay. Conclusion The constructed pSUPER-NRF2-A1、B1 showed an interfering effect on the expression of NRF2 and could product the stable cells with low NRF2 expression. Therefore，the pSUPER vector constitutes a new and powerful system to analyze NRF2 gene function in colon cancer.