Oncogenic viruses frequently target the pathways controlled by tumor suppressor genes, suggesting an extra function for these proteins as antiviral factors. The control exerted by the tumor suppressor Arf on cellular proliferation is crucial to restrict tumor development; however, a potential contribution of Arf to prevent viral infectivity has remained unexplored. In the present study, we investigated the consequences of loss or increased expression of Arf on viral infection. Our results reveal that ARF expression is induced by interferon and after viral infection. Furthermore, we show that ARF protects against viral infection in a gene dosage-dependent manner, and that this antiviral action is mediated in part by PKR through a mechanism that involves ARF-induced release of PKR from nucleophosmin complexes. Finally, Arf-null mice were hypersensitive to viral infection compared to wild-type mice. Together, our results reveal a novel and unexpected role for the tumor suppressor ARF in viral infection surveillance.