The proportion of human isolates with reduced neuraminidase inhibitors (NAIs) susceptibility in highly pathogenic avian influenza (HPAI) H7N9 virus was as high as 13%. These drug-resistant strains showed good replication capacity without serious loss of fitness. At the presence of oseltamivir, R229I substitution were found in HA1 region of the HPAI H7N9 virus before NA R292 K appeared. HPAI H7N9 or H7N9/PR8 recombinant viruses were developed to study whether HA R229I could increase the fitness of the H7N9 virus bearing NA 292 K. Replication efficiency was assessed in MDCK or A549 cells. Neuraminidase enzyme activity and receptor-binding ability were analyzed. The pathogenicity in C57 mice was evaluated. Antigenicity analysis was conducted through a two-way HI test, in which the antiserum was obtained from immunized ferrets. Transcriptomic analysis of MDCK infected with HPAI H7N9 24hpi was done. It turned out that HA R229I substitution from oseltamivir induction in HA1 region increased 1)replication ability in MDCK(P < 0.05) and A549(P < 0.05), 2)neuraminidase enzyme activity, 3)binding ability to both α2,3 and α2,6 receptor, 4)pathogenicity to mice(more weight loss; shorter mean survival day; viral titer in respiratory tract, P < 0.05; Pathological changes in pneumonia), 5) transcriptome response of MDCK, of the H7N9 virus bearing NA 292 K. Besides, HA R229I substitution changed the antigenicity of H7N9/PR8 virus (＞4-fold difference of HI titer). It indicated that through the fine-tuning of the HA-NA balance, R229I increased the fitness and change the antigenicity of a H7N9 virus bearing NA 292 K. Public health attention of this mechanism needs to be drawn.