Although alteration in host cellular translation machinery occurs in virus-infected cells, the role of such alteration and precise pathogenic processes are not well understood. Influenza A virus (IAV) infection shuts off host cell gene expression at transcriptional and translational levels. Here, we found that protein level of eukaryotic translation initiation factor 4B (eIF4B), an integral component of translation initiation apparatus, was dramatically reduced in A549 cells as well as in the lung, spleen and thymus of mice infected with IAV. The decrease in eIF4B level was attributed to lysosomal degradation of eIF4B, which was induced by viral NS1 protein. Silencing eIF4B expression in A549 cells significantly promoted IAV replication, and conversely, overexpression of eIF4B markedly inhibited the viral replication. Importantly, we observed that eIF4B knockdown transgenic mice were more susceptible to IAV infection, exhibiting faster weight loss, shorter survival time, and more severe organ damage. Furthermore, we demonstrated that eIF4B regulated expression of interferon-induced transmembrane protein 3 (IFITM3), a critical protein involved in immune defense against a variety of RNA viruses, including influenza virus. Taken together, our findings reveal that eIF4B plays an important role in host defense against IAV infection at least by regulating the expression of IFITM3 that restricts viral entry and thereby blocks early stages of viral production. These data also indicate that influenza virus has evolved a strategy to overcome host innate immunity by down-regulating eIF4B protein. IMPORTANCE: Influenza A virus (IAV) infection stimulates host innate immune system, in part, by inducing interferons (IFNs). Secreted IFNs activate JAK/STAT pathway, leading to elevated transcription of a large group of IFN-stimulated genes that have antiviral function. To circumvent the host innate immune response, influenza virus has evolved multiple strategies for suppressing the production of IFNs. Here, we show that IAV infection induces lysosomal degradation of eIF4B protein; and eIF4B inhibits IAV replication by upregulating expression of interferon-induced transmembrane protein 3 (IFITM3), a key protein that protects the host from virus infection. Our finding illustrates a critical role of eIF4B in host innate immune response, and provides novel insights into complex mechanisms by which influenza virus interacts with its host.