Avian influenza A viruses are serious veterinary pathogens that normally circulate among avian populations, causing substantial economic impacts. Some strains of avian influenza A viruses, such as H5N1, H9N2, and recently reported H7N9, have been occasionally found to adapt to humans from other species. In order to replicate efficiently in the new host, influenza viruses have to interact with a variety of host factors. In the present study, H7N9 nucleoprotein was transfected into human HEK293T cells, followed by immunoprecipitated and analyzed by proteomics approaches. A series of host proteins co-immunoprecipitated were identified with high confidence, some of which were found to be acetylated at their lysine residues. Bioinformatics analysis revealed that spliceosome might be the most relevant pathway involved in host response to nucleoprotein expression, increasing our emerging knowledge of host proteins that might be involved in influenza virus replication activities.