H9N2 subtype, a low pathogenic avian influenza virus, is emerging as a major causative agent circulating poultry workplaces across China and other Asian countries. Increasing case number of interspecies transmissions to mammals reported recently provoked a great concern about its risks inducing global pandemics. In an attempt to understand the underlying mechanism of how the H9N2 virus disrupts the interspecies segregation to transmit to mammals. A mutant H9N2 strain was obtained by passaging the wildtype H9N2 A/chicken/Hong Kong/G9/1997 eight times from lung to lung in BALB/c mice. Our finding revealed that mice manifested severe clinical symptoms including losses of body weight, pathological damages in pulmonary sites and all died within two weeks after infected with the mutated H9N2, whereas all mice survived upon infected with wildtype strain in comparison, which suggested increased pathogenicity of the mutant strain. In addition, mice showed enhanced levels of proinflammatory cytokines in sera, including IL-6, TNF-α and IL-1β compared to those subjected to wildtype viral infections. Sequence analysis showed that five amino acid substitutions occurred at PB2627, HA87, HA234, NP387 and M156, and a deletion mutation happened in the M gene (M157). Of these mutations, PB2 E627K played key roles in modulating lethality in mice. Taken together, the mutant H9N2 strain obtained by serial passaging of its wildtype in mice significantly increased its virulence leading to death of mice, which might be associated the accumulated mutations occurred on its genome.