Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by a novel tick-borne phlebitis virus of Bunyaviridae, newly named as Banda virus, characterized by high fever, thrombocytopenia, and leukopenia with high case fatality. Currently, no specific antiviral drugs are available to treat patients. Here, we report a natural lipid metabolite 25-Hydroxycholesterol (25HC) that inhibited SFTSV entry by activating the activity of acetyl-CoA-cholesterol acyltransferase (ACAT), a lipid metabolism enzyme, affecting the accessible cholesterol translocation between the cytoplasm and plasma membranes (PMs). We also found that 25HC could inhibit the extracellular vesicles carrying virions into target cells, thus preventing the vesicles from mediating virus spread. In short, 25HC reduced the PMs-accessible cholesterol through modulating lipid metabolism, thereby inhibiting the double infection of cells by virions and infectious EVs, and potentially reducing the efficiency of SFTSV dissemination.