Itraconazole (ITZ) is a well-known antifungal agent that also has anticancer activity. In this study, we identify ITZ as a broad-spectrum inhibitor of enteroviruses (e.g., poliovirus, coxsackievirus, enterovirus-71, rhinovirus). We demonstrate that ITZ inhibits viral RNA replication by targeting oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4). Consistently, OSW-1, a specific OSBP/ORP4 antagonist, also inhibits enterovirus replication. Knockdown of OSBP inhibits virus replication, whereas overexpression of OSBP or ORP4 counteracts the antiviral effects of ITZ and OSW-1. ITZ binds OSBP and inhibits its function, i.e., shuttling of cholesterol and phosphatidylinositol-4-phosphate between membranes, thereby likely perturbing the virus-induced membrane alterations essential for viral replication organelle formation. ITZ also inhibits hepatitis C virus replication, which also relies on OSBP. Together, these data implicate OSBP/ORP4 as molecular targets of ITZ and point to an essential role of OSBP/ORP4-mediated lipid exchange in virus replication that can be targeted by antiviral drugs. Display omitted •ITZ, an antifungal and anticancer agent, is a broad-spectrum enterovirus inhibitor•OSBP and ORP4 are identified as novel targets of ITZ•ITZ binds OSBP and inhibits OSBP-mediated lipid exchange at membrane contact sites•ITZ also inhibits hepatitis C virus replication Strating et al. present the antifungal drug itraconazole as a novel inhibitor of a broad range of viruses, including poliovirus and hepatitis C virus. Itraconazole acted on a novel target, the oxysterol-binding protein (OSBP), a protein that has an essential role in lipid transfer.