Influenza A virus infection is a major global health concern causing significant mortality, morbidity, and economic loss. Antiviral chemotherapeutics that target influenza A virus are available; however, rapid emergence of drug-resistant strains has been reported. Consequently, there is a burgeoning need to identify novel anti-influenza A drugs, particularly those that target host gene products required for virus replication, to reduce the likelihood of drug resistance. In this study, a small interfering RNA (siRNA) screen was performed to identify host druggable gene targets for anti-influenza A virus therapy. The host organic anion transporter-3 gene (OAT3), a member of the SLC22 family of transporters, was validated as being required to support influenza A virus replication. Probenecid, a prototypical uricosuric agent and chemical inhibitor of organic anion transporters known to target OAT3, was shown to be effective in limiting influenza A virus infection in vitro (50% inhibitory concentration [IC(50)] of 5.0 × 10(-5) to 5.0 × 10(-4) μM; P < 0.005) and in vivo (P < 0.05). Probenecid is widely used for treatment of gout and related hyperuricemic disorders, has been extensively studied for pharmacokinetics and safety, and represents an excellent candidate for drug repositioning as a novel anti-influenza A chemotherapeutic.