[Abtract] Objective In order to explore the pathogenity molecular basis of A/H6N1 AIV .we established A/H6N1 subtype avian influenza virus reverse genetic System and evaluation of the pathogenicity of PB2 E627K in A/H6N1 Avian InfluenzaVirus. Methods rA/H6N1 and rA/H6N1-627 were rescued by A/Mallard/SanJiang / 275/2007 strain reverse genetic system and site mutation technology. 101EID50--106EID50 rA/H6N1 and rA/H6N1-627 inoculated mice separately. the virology data were collected by body weight change, MLD50 and vius titration. Results A/H6N1 subtype avian influenza virus reverse genetic System was established successfully , Sequence analysis results show that the 8 gene segments of rA/H6N1 are derived from the genome of A/H6N1and have no nucleic acid change. rA/H6N1 can infect mice efficiently but are not lethal for mice , showing low pathogenicity in mice (MLD50 >106.5EID50). The distribution of virus and the virus titer shows no difference with A/H6N1. so the biological characteristics keep the same with A/H6N1. rA/H6N1-627 can infect mice, with the decreasing weight, but are not lethal for all the mice in 106EID50 group. Conclusion The results show that PB2-E627K which playing important roles in H5N1 is not the determining factor in A/H6N1.The reverse genetic system and site mutation technology develop the foundations in pathogenic mechanism, transmission mechanism and virus gene function of A/H6N1 AIV , also open up new ways for the A/H6N1 AIV vaccines.