Abstract: 【Abstract】 Objective To construct the sh-\rpS6 lentivirus vector targeting ribosomal protein S6 (rpS6) and explore its effect on proliferation in lung adenocarcinoma A549 cell lines. Methods Sequences targeting the \rpS6 gene were selected. The double strand shRNA oligo was ligated to pGCsil-\GFP lentivirus vector and transformed into \E.coli. The resulting recombinant vector was verified by sequencing. After transfection and lentivirus packing, the viral particles were collected and infected A549 cells. After selection of GFP positive cells by FACS, mRNA and protein expression levels of rpS6 were determined by real time PCR and Western blot. In the following experiment, the proliferation changes of A549 cell lines after the interference by sh-\rpS6 was investigated by using CCK-8 kit. Results The sequencing result confirmed that pGCsil-sh-\rpS6-GFP vector was successfully developed. Stably transfected A549 cell lines by sh-\rpS6 were selected through FACS, with a selection ratio of 86.80%. The silencing effects of sh-rpS6 were determined by real time PCR and Western blot, suggesting that mRNA and protein expression of rpS6 in the targeted cells reduced by (79.72±6.83)% and （83.77±12.13)%, significantly lower than those of control groups. \In vitro showed the cell proliferation with sh-\rpS6 was significantly slower than that of controls (\P<0.05). Conclusion The constructed sh-\rpS6 lentivirus vector could inhibit the expression of rpS6 in A549 cell lines effectively and significantly slow the cell proliferation \In vitro