Influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but the mechanism is not clear. Here, it is reported that influenza A virus NS1 protein is responsible for PI3K/Akt pathway activation. It was demonstrated that the NS1 protein interacts with the p85 regulatory subunit of PI3K via direct binding to the SH3 and C-terminal SH2 domains of p85. Consensus binding motifs for SH3 and SH2 domains were found in influenza A virus NS1, namely an SH2-binding motif (YXXXM) at aa 89, SH3-binding motif 1 (PXXP) around aa 164 and SH3-binding motif 2 around aa 212. Mutant virus encoding NS1 protein with mutations in the SH-binding motifs failed to interact with SH domains of p85 and did not activate the PI3K/Akt pathway. The mutant virus is attenuated in Madin-Darby canine kidney cells. Our study has established a novel function of NS1: by interacting with p85 via the SH-binding motifs, NS1 can activate the PI3K/Akt pathway.