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Objective To investigate the effects of NOK added DFG motif on proliferation and G1/S distribution of NIH3T3 cells. Methods SWISS MODEL homologous modeling was used to confirm the location of DFG addition; we used CCK-8 and MTT methods to measure the cell proliferation of NIH3T3 transiently transfected pcDNA3.0 or pcDNA3.0-DFG-NOK; Flow cytometry was used to measure cell cycle; Western-blot was used to exam the phosphorylation of Akt,Erk,STAT1,STAT3,JAK3 and the expression of cyclin D1. Results Compared with the pcDNA3.0 transient transfection: 1)The proliferation of pcDNA3.0-DFG-NOK over-expression NIH3T3 cells was inhibited. 2)The entrance of G1/S in NIH3T3 cells transiently transfected pcDNA3.0-DFG-NOK was obstructed. 3)Transient transfection of pcDNA3.0-DFG-NOK could inhibit phosphorylation of Erk,STAT1,STAT3,JAK3 and expression of CyclinD1. Conclusions DFG-NOK inhibits proliferation of NIH3T3 cells and obstructs cell cycle G1 phase entering into S phase.