Influenza A virus (IAV) is a significant zoonotic pathogen that poses a considerable challenge to public health due to its continuous mutations. Lipid droplets (LDs) have been shown to play an important role in the process of several viral infections. However, their role in IAV infection remains unclear. Here, we found that IAV infection altered the lipid metabolism and increased the content of LDs in the lungs of mice. In vitro, IAV infection also mediated the formation of LDs in A549 cells. Besides, inhibition of the formation of lipid droplets can significantly suppress IAV replication and the release of inflammatory factors, indicating that LDs could facilitate the virus replication and inflammatory response. Furthermore, we discovered that IAV infection could activate the SREBP2, a crucial lipid-regulating transcription factor that regulates the expressions of downstream proteins named HMGCR and HMGCS. HMGCR and HMGCS involved in the process of cholesterol synthesis, which further promoted the formation of LDs. Additionally, the use of fatostatin that specifically inhibits the maturation of SREBP2 was able to significantly suppress the viral replication of H5N1 in cells and effectively ameliorated IAV-induced lung injury in mice, which eventually promoted the survival rate of infected mice. Taken together, we demonstrate the essential roles of lipid metabolism and LD formation in IAV replication and pathogenesis, which may better facilitate the advancement of new strategies against IAV infection, especially the highly pathogenic H5N1 virus.