Avian influenza caused by H9N2 subtype avian influenza virus (AIV) poses a great threat to the healthy development of the poultry industry. Vimentin is closely related to intracellular lipid metabolism, which plays an important role during the viral infection process. However, the function of lipid metabolism and vimentin on H9N2 AIV replication is unclear. In this paper, the cholesterol level and 3-hydroxy-3-methylglutaryl coenzyme a reductase (HMGCR) phosphorylation were investigated in vimentin knockout (KO) and human cervical carcinoma cells (HeLa) cell with or without AIV infection. The results showed that compared to the control group without infected with H9N2 subtype AIV, the cholesterol contents were significantly increased, while HMGCR phosphorylation level was reduced in both KO and HeLa cell after virus infection. Furthermore, viral replication was significantly inhibited in the cells treated with the cholesterol inhibitor lovastatin. Compared with the control group, adenylate activated protein kinase (AMPK), a kinase regulating HMGCR enzymatic activity was inhibited in both KO and HeLa cells in the infected virus group, and AMPK phosphorylation levels were significantly lower in KO HeLa cell than that of HeLa cells. Additionally, after MβCD treatment, viral hemagglutinin (HA) gene level was significantly decreased in HeLa cells, while it was significantly increased in KO HeLa cells. In addition, vimentin expression was significantly increased in MβCD-treated HeLa cells with the viral infection and returned to normal levels after exogenous cholesterol to backfill the MβCD-treated cells. Therefore, the disruption of lipid rafts during the binding phase of viral invasion of cells significantly reduced viral infection. These studies indicated that the lipid rafts and cholesterol levels might be critical for H9N2 subtype AIV infection of human-derived cells and that vimentin might play an important role in the regulation of lipids on viral replication, which provided an important antiviral target against influenza virus.