Here we report that epidermal growth factor (EGF) inhibits the replication of vesicular stomatitis virus (VSV) in human epidermoid carcinoma A-431 cells, as measured by reduction in infectious viral particle production, cell protection assay, and inhibition of viral protein synthesis. The observed antiviral effects of EGF were not apparently due to inhibitory effects on the initial events in VSV infection as postinfection treatment of cells with EGF also resulted in significant inhibition in the levels of viral proteins. The EGF-mediated inhibition of VSV protein synthesis was specific in nature as prior treatment of cells with an excess of anti-EGF receptor monoclonal antibody, which blocks EGF binding, reduced the inhibition of VSV proteins elicited by EGF. In summary, the observed antiviral action of EGF was an early effect of EGF and was mediated via an EGF-responsive cellular pathway(s) that could impair VSV replication.