With the prevalence of novel influenza strain and drug resistance virus, it is urgent to develop effective and low-toxicity anti-influenza therapeutics. Here we found that pterostilbene effectively inhibited influenza A virus infection. Mechanism study demonstrated that pterostilbene inhibited virus infection through interaction with influenza non-structural protein 1 (NS1), in turn promoted the induction of antiviral type I interferon and impeded the pro-viral PI3K/Akt pathway activation during influenza virus infection, inducing a synergistic inhibitory effect against influenza virus infection. Thus, pterostilbene can be a promising anti-influenza agent for the future antiviral drug exploitation.