Objective:To investigate the alteration of cell cycle arrest of 5-FU to Panc-1 cell line after downregulation of hENT1 (human equilibrative nucleoside transporter 1) mRNA expression by RNAi. Methods:The plasmid(pSilence-hENT1 4.1-CMV neo)which expresses shRNA(small hairpin RNA) specifically for human equilibrative nucleoside transporter 1(hENT1) was designed and recombined. Then, pSilence-hENT1 4.1-CMV neo was transfected into Panc-1 cells with Lipofectamin 2000. After that, the transfected cells(pSilence-hENT1 Panc-1) was selected with medium containing G418(600 -滋g/ml). hENT1-mRNA expression was detected by RT-PCR to determine whether it was downregulated efficiently and stably by RNAi. Cells were cultured in the medium with 20 μmol/L of 5-FU 24 h. Then the cell cycle was detected by flow cytometry(FCM). Results:pSilence-hENT1 4.1-CMV neo sequencing showed that the designed DNA fragment was successfully inserted into pSilence 4.1-CMV neo plasmid. The downregulation of hENT1-mRNA expression was found and it had maintained for 2 months(15 passages) in pSilence-hENT1 Panc-1 cell. The percentage of G0/G1 phase cells in pSilence-hENT1 Panc-1 group was increased to 120%(P < 0.01) of the Panc-1 group,and the percentage of S phase cells was reduced to 66.76%(P < 0.01) compared with Panc-1 group. Conclusion:The downregulation of hENT1-mRNA expression by RNAi enhances G0/G1 phase arrest of 5-FU to pancreatic cancer cells.