Infection and replication of enveloped viruses require host cells to supply substantial amounts of cellular cholesterol for processes such as binding, entry, trafficking, assembly, and budding. However, the mechanisms by which influenza A virus (IAV) regulates cholesterol biosynthesis remain poorly understood. In this study, we demonstrate that IAV infection induces the expression of the retinoic acid-related orphan receptor γ (RORγ), an orphan nuclear receptor, which cooperates with the sterol regulatory element-binding protein-2 (SREBP2) to regulate the expression of the 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase (HMGCR), a key enzyme in cholesterol biosynthesis. RORγ knockout and treatment with two RORγ inhibitors, XY018 and GSK805, suppress IAV-induced HMGCR expression, cholesterol biosynthesis, and viral replication. Notably, exogenous cholesterol rescues the inhibitory effect of XY018 on viral replication. Mechanistically, we show that IAV infection activates RORγ expression through the TGF-β-activated kinase 1 (TAK1) and its downstream kinases, the c-Jun N-terminal kinase (JNK) and the IκB kinase (IKK), which in turn activate AP1 and NF-κB. In vivo, RORγ knockout reduces IAV replication, alleviates body weight loss, and prolongs survival in infected mice. Furthermore, XY018 treatment reduces both viral replication and inflammation in the lungs of IAV-infected mice. Our findings provide novel mechanistic insights into how IAV infection upregulates cholesterol biosynthesis to facilitate viral replication.