Influenza A virus infection stimulates a wide range of virus-supportive or antiviral mechanisms in host cells. p21-Activated kinase 1 (PAK1) is a serine/threonine kinase that regulates a number of fundamental cellular processes and has been implicated in the modulation of virus replication. Here, we investigated the role of PAK1 activation during influenza A virus infection and found that virus propagation corresponded to stimulated PAK1 phosphorylation. Moreover, transfection of the active form of PAK1 (PAK1-T423E) in A549 cells induced higher viral titers (∼10-fold differences) compared to that in the control vector or inactive PAK1 (PAK1-K299R)-transfected cells. PAK1-specific siRNA knockdown also resulted in 10-100-fold reductions in virus yields compared to that in the control siRNA-treatment (p<0.05). We further showed that treatment with PAK18, a PAK1 peptide inhibitor, resulted in marked suppression of both ERK 1/2 phosphorylation and infectious virus production, which was comparable to that by U0126, a specific MEK/ERK inhibitor. These results provide evidence for the importance of PAK1 activation during influenza virus infection and its association with ERK in regulating virus replication. The present study also implicates PAK1 as a potential therapeutic target for managing influenza virus infections.