Elimination of infected cells by programmed cell death is a well-recognized host defense mechanism to control the spread of infection. In addition to apoptosis, necroptosis is also one of the mechanisms of cell death that can be activated by viral infection. Activation of necroptosis leads to the phosphorylation of mixed-lineage kinase domain-like protein (MLKL) by receptor interacting protein kinase (RIPK)-3 and results in MLKL oligomerization and membrane translocation, leading to membrane disruption and loss of cellular ion homeostasis. It has recently been reported that influenza A virus (IAV) infection induces necroptosis. However, the underlying mechanism of IAV-mediated necroptosis process, particularly the roles of IAV proteins in necroptosis, remains unexplored. Here, we report that IAV infection induces necroptosis in macrophages and epithelial cells. We demonstrate that the NS1 protein of IAV interacts with MLKL. The coiled-coil domain 2 of MLKL has a predominant role in mediating MLKL interaction with NS1. Interaction of NS1 with MLKL increases MLKL oligomerization and membrane translocation. Moreover, the MLKL-NS1 interaction enhances MLKL-mediated NLRP3 inflammasome activation, leading to increased IL-1β processing and secretion.IMPORTANCE Necroptosis is a programmed cell death that is inflammatory in nature owing to the release of danger-associated molecular patterns from the ruptured cell membrane. However, necroptosis also constitutes an important arm of host immune responses. Thus, a balanced inflammatory response determines the disease outcome. We report that the NS1 protein of IAV participates in necroptosis by interacting with MLKL, resulting in increased MLKL oligomerization and membrane translocation. These results reveal a novel function of the NS1 protein and the mechanism by which IAV induces necroptosis. Moreover, we show that this interaction enhances the NLRP3 inflammasome activation and IL-1β processing and secretion. The information may contribute to a better understanding of the role of necroptosis in IAV-induced inflammation.