Infection by RNA virus induces oxidative stress in host cells. Accumulating evidence suggests that cellular redox status plays an important role in regulating viral replication and infectivity. In this study, experiments were performed to determine whether the thiol antioxidant glutathione (GSH) blocked influenza viral infection in cultures of Madin-Darby canine kidney cells or human small airway epithelial cells. Protection against production of active virus particles was observed at a low (0.05-0.1) multiplicity of infection (MOI). GSH inhibited expression of viral matrix protein and inhibited virally induced caspase activation and Fas upregulation. In BALB/c mice, inclusion of GSH in the drinking water decreased viral titer in both lung and trachea homogenates 4 d after intranasal inoculation with a mouse-adapted influenza strain A/X-31. Together, the data suggest that the thiol antioxidant GSH has an anti-influenza activity in vitro and in vivo. Oxidative stress or other conditions that deplete GSH in the epithelium of the oral, nasal, and upper airway may, therefore, enhance susceptibility to influenza infection.