The NS1 protein of influenza A virus (IAV) is a multi-functional protein which can antagonize host immune system and facilitate viral replication by interacting with host factors. However, the novel partners in host cells interacting with NS1 need to be fully elucidated. In the current study, we identified hnRNPH1 as a novel binding partner of NS1 to regulate IAV replication. Notably, overexpression of hnRNPH1 decreased IAV multiplication, while knockdown of hnRNPH1 enhanced IAV replication. hnRNPH1 can interact with NS1 to change the intracellular localization and splicing function of NS1, and impact IAV replication through interacting with p53 to regulate cell apoptosis. In addition, the RBD domain of NS1 and the RRM and NLS regions of hnRNPH1 may be the major sites for their interaction. In summary, our studies identified hnRNPH1 as a novel NS1-binding protein and elucidated its regulatory roles in IAV replication, which will provide new insights into the roles of NS1 binding proteins, and give a reference for anti-IAV therapy based on NS1-host interaction.