Influenza　A　virus　(IAV)　is　a　major　human　pathogen　with　the　potential　to　become　pandemic.　IAV　contains　only　eight　RNA　segments;　thus,　the　virus　must　fully　exploit　the　host　cellular　machinery　to　facilitate　its　own　replication.　In　an　effort　to　comprehensively　characterize　the　host　machinery　taken　over　by　IAV　in　mammalian　cells,　we　generated　stable　A549　cell　lines　with　over-expression　of　the　viral　non-structural　protein　(NS1)　to　investigate　the　potential　host　factors　that　might　be　modulated　by　the　NS1　protein.　We　found　that　the　viral　NS1　protein　directly　interacted　with　cellular　Rac1　and　facilitated　viral　replication.　Further　research　revealed　that　NS1　down-regulated　Rac1　activity　via　post-translational　modifications.　Therefore,　our　results　demonstrated　that　IAV　blocked　Rac1-mediated　host　cell　signal　transduction　through　the　NS1　protein　to　facilitate　its　own　replication.　Our　findings　provide　a　novel　insight　into　the　mechanism　of　IAV　replication　and　indicate　new　avenues　for　the　development　of　potential　therapeutic　targets.