H9N2 influenza viruses continue to circulate in wild birds and poultry in Eurasian countries and have repeatedly infected mammals, including pigs and humans, posing a significant threat to public health. To understand the adaptation of H9N2 influenza viruses to mammals, we serially passaged a nonpathogenic duck-origin H9N2 influenza virus, A/duck/Jiangsu/1/2008 (DK1), in mouse lungs. Increased virulence was detectable after five sequential passages, and a highly pathogenic mouse-adapted strain (DK1-MA) with a 50 % mouse lethal dose of 10(2.37) 50 % egg infectious dose was obtained after 18 passages. DK1-MA grew faster and reached significantly higher titers than DK1 in mouse lungs and could sporadically spread to other organs. Moreover, DK1-MA induced a greater magnitude of pulmonary edema and higher levels of inflammatory cellular infiltration in bronchoalveolar lavage fluids than DK1 did. Genomic sequence alignment revealed eight amino acid substitutions (HA-L80F, HA-N193D, NA-A27T, PB2-F404L, PA-D3V, PA-S225R, NP-V105M, M1-A166V) in six viral proteins of DK1-MA compared with DK1 virus. Except for HA-L80F, the other seven substitutions were all located in known functional regions involved in interaction of viral proteins or interaction between the virus and host factors. Taken together, our results suggest that multiple amino acid substitutions may be involved in the adaptation of H9N2 avian influenza virus to mice, resulting in lethal infection, enhanced viral replication, severe pulmonary edema, and excessive inflammatory cellular infiltration in lungs. These observations provide helpful insights into the pathogenic potential of H9N2 avian influenza viruses that could pose threats to human health in the future.