Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that is widespread in cattle. However, only a few studies about the pathogenesis of BoHV-4 primary infection have been reported. In the present study, ex vivo models with bovine nasal and tracheal mucosa explants were used to study the cellular BoHV-4-host interactions. Infection was observed in nasal but not in tracheal epithelial cells. To find a possible correlation between the integrity and restricted infection of the respiratory epithelium, both nasal mucosal and tracheal explants were treated with EGTA, a drug that disrupts the intercellular junctions, before inoculation. The infection was analyzed based on the number of plaques, plaque latitude and number of infected single cells, as determined by immunofluorescence. BoHV-4 infection in nasal mucosal explants was enhanced upon opening the tight junctions with EGTA. Infection in tracheal explants was only found after treatment with EGTA. In addition, primary bovine respiratory epithelial cells (BREC) were isolated, grown at the air-liquid interface and infected either at the apical or basolateral side by BoHV-4. The results showed that BoHV-4 preferentially bound to and entered BREC at the basolateral surfaces of both nasal and tracheal epithelial cells. The percentage of BoHV-4 infection was significantly increased both from nasal and tracheal epithelial cells after treatment with EGTA, which indicates that the BoHV-4 receptor is mainly located at the basolateral surface of these cells. Thus, our findings demonstrate that integrity of the respiratory epithelium is crucial in the host's innate defense against primary BoHV-4 infections.