The repurposing of drugs approved by the regulatory agencies for other indications is emerging as a valuable alternative for the development of new antimicrobial therapies， involving lower risks and costs than the de novo development of novel antimicrobial drugs. Adenovirus infections have showed a steady increment in recent years， with a high clinical impact in both immunosuppressed and immunocompetent patients. In this context， the lack of a specific drug to treat these infections supports the search for new therapeutic alternatives. In this study， we examined the anti-HAdV properties of mifepristone， a commercially available synthetic steroid drug. Mifepristone showed significant in vitro anti-HAdV activity at low micromolar concentrations with little cytotoxicity. Our mechanistic assays suggest that this drug could affect the microtubule transport， interfering with the entry of the virus into the nucleus and therefore inhibiting HAdV infection.