Viruses exploit host cell machinery to support their replication. Defining the cellular proteins and processes required for a virus during infection is crucial to understanding the mechanisms of virally induced disease and designing host-directed therapeutics. Here, we perform a genome-wide CRISPR-Cas9-based screening in lung epithelial cells infected with the PR/8/NS1-GFP virus and use GFPhi cell as a unique screening marker to identify host factors that inhibit influenza A virus (IAV) infection. We discovered that APOE affects influenza virus infection both in vitro and in vivo. Cell deficiency in APOE conferred substantially increased susceptibility to IAV; mice deficient in APOE manifested more severe lung pathology, increased virus load, and decreased survival rate. Mechanistically, lack of cell-produced APOE results in impaired cell cholesterol homeostasis, enhancing influenza virus attachment. Thus, we identified a previously unrecognized role of APOE in restraining IAV infection.