ABSTRACTThe World Health Organization declared the spread of monkeypox virus (MPXV) across multiple African countries a public health emergency of international concern in August 2024. This marks the second such declaration in the past 2 years in response to the MPXV transmission. The re‐emergence of the MPXV 3 years after the start of the SARS‐CoV‐2 epidemic further emphasizes the need to develop broad spectrum antivirals that might control the spread of poorly understood pathogens. The induction of innate immune responses to a viral infection triggers rapid expression of type‐I‐interferon, which subsequently leads to the differential regulation of over 300 genes that collectively establish an antiviral state. While most of these “interferon‐stimulated genes” (ISGs) are upregulated, some are downregulated. Notably, the expression of certain ISGs involved in lipid metabolism is suppressed, which can be exploited by viruses to facilitate their replication. Herein, we report that the expression of fatty acid synthase (FASN), an enzyme involved in de novo biosynthesis of fatty acids, was significantly reduced upon MPXV infection. Moreover, MPXV infection was impaired in FASN knockout cells, and biological inhibitors of FASN significantly inhibited MPXV. Interestingly, chosterol‐25‐hydroxylase was induced in MPXV‐infected cells, and its enzymatic product, 25‐hydroxychlosterol (25HC), blocked MPXV infection. Overall, this study suggests that 25HC and FASN inhibitors exhibit significant antiviral activity and may have therapeutic applications in combating understudied infectious diseases in early outbreak settings when targeted therapies have not yet been developed.