Variola virus, the causative agent of smallpox, enters and exits the host via the respiratory route. To better understand the pathogenesis of poxvirus infection and its interaction with respiratory epithelia, we used vaccinia virus and examined its interaction with primary cultures of well-differentiated human airway epithelia. We found that vaccinia virus preferentially infected the epithelia through the basolateral membrane and released viral progeny across the apical membrane. Despite infection and virus production, epithelia retained tight junctions, transepithelial electrical conductance, and a steep transepithelial concentration gradient of virus, indicating integrity of the epithelial barrier. In fact, during the first four days of infection, epithelial height and cell number increased. These morphological changes and maintenance of epithelial integrity required vaccinia virus growth factor, which was released basolaterally, where it activated epidermal growth factor 1 receptors. These data suggest a complex interaction between the virus and differentiated airway epithelia; the virus preferentially enters the cells basolaterally, exits apically, and maintains epithelial integrity by stimulating growth factor receptors.