Cholesterol-24-hydroxylase (CH24H or Cyp46a1) is a reticulum-associated membrane protein that plays an irreplaceable role in cholesterol metabolism in the brain and has been well-studied in several neuro-associated diseases in recent years. In the present study, we found that CH24H expression can be induced by several neuroinvasive viruses, including vesicular stomatitis virus (VSV), rabies virus (RABV), Semliki Forest virus (SFV) and murine hepatitis virus (MHV). The CH24H metabolite, 24-hydroxycholesterol (24HC), also shows competence in inhibiting the replication of multiple viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 24HC can increase the cholesterol concentration in multivesicular body (MVB)/late endosome (LE) by disrupting the interaction between OSBP and VAPA, resulting in viral particles being trapped in MVB/LE, ultimately compromising VSV and RABV entry into host cells. These findings provide the first evidence that brain cholesterol oxidation products may play a critical role in viral infection.